PCSK9-targeting peptide immunogens and formulations thereof for preventing and treating PCSK9-mediated disorders

Abstract

The present disclosure is directed to peptide immunogenic constructs targeting the catalytic domain of PCSK9 protein, compositions comprising the constructs, antibodies induced by the constructs, and methods for preparing and using the constructs and compositions thereof. The peptide immunogenic constructs of the present disclosure have more than about 20 amino acids and include (a) a B cell epitope having more than about 7 consecutive amino acid residues derived from the PCSK9 and LDL-R receptor-binding region of the catalytic domain of PCSK9 protein, (b) a heterologous Th epitope, and (c) an optional heterologous spacer. The PCSK9 peptide immunogenic constructs of the present disclosure stimulate the production of highly specific antibodies directed against the PCSK9 site that binds to LDL-R, enabling the prevention and / or treatment of patients with PCSK9-mediated disorders, including elevated serum levels of low-density lipoprotein cholesterol (LDL-C) and CV events.

Description

[Technical Field]

[0001] This application is a PCT international application claiming the benefit of U.S. Provisional Application No. 62 / 966,645, filed January 28, 2020, which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to peptide immunogen constructs and formulations thereof that target proprotein convertase subtilisin-kexin type 9 (PCSK9) for the prevention and treatment of patients with PCSK9-mediated disorders, including elevated serum levels of low-density lipoprotein cholesterol (LDL-C) and cardiovascular events. [Background technology]

[0003] Cardiovascular (CV) disease is the leading cause of death worldwide, accounting for an estimated 31.5% of all deaths in 2013. The burden of atherosclerotic CV disease (ASCVD) is particularly high, which can manifest as coronary heart disease (CHD), cerebrovascular disease, and peripheral arterial disease. Increased serum levels of low-density lipoprotein cholesterol (LDL-C) are an independent risk factor for ASCVD, and clinical trial data have demonstrated an association between lowering LDL-C and reduced CV risk. Consequently, reducing LDL-C is an important strategy for primary and secondary prevention of ASCVD.

[0004] The cornerstone of LDL-C lowering is statins, which inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. Large randomized trials have demonstrated the efficacy of statins in reducing the risk of major vascular events by approximately 25% per mmol / L reduction in LDL-C per year after 1 year of statin use, with the absolute benefit of statin therapy being greater in higher-risk patients. In contrast, nonstatin lipid-lowering therapies, such as niacin and cholesteryl ester transfer protein inhibitors, have shown no CV benefit or may even increase the risk of CV events and death. Combination therapy with statins and ezetimibe to lower LDL-C has shown modest improvements in CV outcomes in patients with acute coronary syndromes and suggests additional clinical benefit by reducing LDL-C to levels below 70 mg / dL, the target of previous clinical practice guidelines. Despite the proven efficacy of statins in reducing LDL-C levels and CV events, additional therapies are needed. Despite statin therapy, some patients remain at high CV risk due to insufficient reduction in LDL-C levels or persistent dyslipidemia unrelated to LDL-C. Furthermore, adverse effects, including myopathy (ranging from mild muscle pain to severe rhabdomyolysis), new or worsening diabetes, and possibly hemorrhagic stroke, may limit the use of statins or the ability to achieve target effective statin doses in certain patients. Recently, another class of drugs that inhibit PCSK9 has been developed for the treatment of hyperlipidemia.

[0005] PCSK9 was first identified in 2001 when elevated protein levels were observed in a study of cerebellar neuron apoptosis. The protein was initially named neuronal apoptosis-regulating converting enzyme 1, and its gene was characterized in 2003. The gene for PCSK9 is located on human chromosome 1p32 and encodes a 692-amino acid serine protease. PCSK9 (GenBank accession number: EAX06660) has the amino acid sequence of SEQ ID NO: 1, as shown in both Table 1 and Figure 2. PCSK9 is primarily expressed in the liver, although lower levels of protein expression have also been found in the intestine, kidney, and central nervous system. Transcription of PCSK9 is primarily regulated by sterol regulatory element-binding protein-2 (SREBP-2). In hepatocytes, PCSK9 is synthesized as a zymogen that requires activation by a separate enzyme and is composed of a signal peptide (residues 1–30), a prodomain (residues 31–152), a catalytic domain (residues 153–454), and a C-terminal (CT) domain (residues 455–692) (Holla, OL, et al., 2011). In the endoplasmic reticulum, PCSK9 undergoes cotranslational autocatalytic cleavage of its signal peptide to produce the prodomain and mature protein, the latter of which is required for secretion from the endoplasmic reticulum to the Golgi apparatus. A characteristic feature of PCSK9 is that the prodomain remains associated with the mature protein, promoting protein folding and preventing enzymatic activity by blocking access to the catalytic site, thereby chaperoning PCSK9 through the secretory pathway.

[0006] PCSK9 circulates in plasma and binds to the cell surface low-density lipoprotein receptor (LDL-R), where it is internalized and subsequently targets the receptor for lysosomal degradation. Binding of PCSK9 to the epidermal growth factor precursor homology domain A (EGF-A) repeats of the LDL-R is mediated by a patch of residues on the catalytic domain of PCSK9. Figure 3 identifies the amino acid residues on PCSK9 and the LDL-R that bind to each other. The catalytic domain of PCSK9 is involved in both autocatalytic cleavage and binding of PCSK9 to the LDL-R.

[0007] Concurrent studies in patients with familial hypercholesterolemia provided insight into the clinical importance of PCSK9, where gain-of-function mutations cause hypercholesterolemia. Mouse models overexpressing PCSK9 demonstrated increased levels of total cholesterol and non-high-density lipoprotein cholesterol (non-HDL-C) and decreased levels of hepatic LDL-R, supporting a causal role for gain-of-function PCSK9 mutations in humans displaying a hypercholesterolemic phenotype. Patients with loss-of-function PCSK9 mutations and associated hypocholesterolemia have been shown to have a reduced risk of CV disease.

[0008] PCSK9 plays a key role in LDL-C metabolism, as described by Chaudhary, R., et al. (2017). LDL-C bound to LDL-R is internalized into hepatocytes via clathrin-coated vesicles, after which the acidic environment of the endosome dissociates LDL-C from its receptor. While recycling vesicles return LDL-R to the cell surface, endosomes containing LDL-C particles fuse with lysosomes, resulting in LDL-C degradation, cholesterol ester hydrolysis, and distribution of free cholesterol to other cellular compartments. At the plasma membrane of hepatocytes, the catalytic domain of secreted PCSK9 associates with LDL-R, is internalized, and enters the endosomal pathway. The low pH of the endosome enhances the affinity of PCSK9 for LDL-R, preventing the receptor from recycling to the cell surface. Instead, the complex is directed to lysosomes, where both components are degraded. In addition, PCSK9 may enhance intracellular LDL-R degradation before secretion, as it can complex with LDL-R in the Golgi apparatus and target the receptor to lysosomes for degradation instead of transport to the plasma membrane.

[0009] Several strategies for PCSK9 inhibition are currently under investigation. The first approach involves interfering with the binding of PCSK9 to LDL-R. Examples of this approach include monoclonal antibodies. Monoclonal antibody therapeutics include evolocumab (REPATHA®), alirocumab (PRALUENT®), and bococizumab. These monoclonal antibodies bind to the catalytic and prodomains of PCSK9, blocking its interaction with LDL-R and neutralizing PCSK9 activity. Studies have shown that maximal suppression of circulating unbound PCSK9 occurs within 4 to 8 hours of monoclonal antibody administration, resulting in a reduction of LDL-C levels of approximately 65% ​​in healthy subjects and approximately 60 to 80% in patients with hypercholesterolemia. PCSK9 inhibition can significantly reduce LDL-C levels in humans, even in patients with a history of statin therapy. Patient populations studied in clinical trials range from those with low CV risk to those with homozygous familial hypercholesterolemia (HoFH), a population of individuals at very high CV risk. While such monoclonal anti-PCSK9 antibodies may prove effective in immunotherapy of PCSK-9-mediated disorders, they are expensive and require monthly administration to sustain sufficient suppression of LDL-C serum levels and thereby achieve sustained clinical benefit. Two review articles (Hess, C., et al., 2018 and Chaudhary, R., et al., 2017) cite additional relevant documents regarding the topics discussed in the Background section above, and these reviews are incorporated herein by reference in their entirety.

[0010] Cost-effective immunotherapeutic treatments targeting the PCSK9 molecule via a safe and well-tolerated vaccination approach remain an exciting and novel intervention and development for PCSK9-mediated disorders. Several approaches along this line of inquiry have been explored, including those by Brunner, S., et al. (U.S. Patent No. 9,669,079) and Champion, R., et al. (U.S. Patent No. 9,987,341), the entire disclosures of which are incorporated herein by reference.

[0011] Conventional epitope-based vaccines suffer from several disadvantages and shortcomings: immunogen preparation methods involve complex chemical conjugation procedures and use expensive pharmaceutical-grade KLH or toxoid proteins as T helper cell carriers, and most of the antibodies induced by such immunogen preparations are directed against the carrier protein(s) and not against the target B cell epitope(s).

[0012] Given the economic and practical disadvantages associated with monoclonal anti-PCSK9 therapy and complex chemical conjugation procedures for peptide / hapten-carrier protein immunogen preparations, there is a clear unmet need to develop immunotherapeutic compositions capable of eliciting a highly specific immune response against functional site(s) on PCSK9, which can be easily administered to patients, can be manufactured cost-effectively under strict Good Manufacturing Practice (GMP), and are effective for global application in the treatment of patients suffering from PCSK9-mediated disorders, including elevated serum levels of LDL-C and CV events.

[0013] References: 1. CHANG, JCC, et al., “Adjuvant activity of incomplete Freund's adjuvant.” Advanced Drug Delivery Reviews, 32(3):173-186 (1998) 1.CHAUDHARY, R., et al., “PCSK9 inhibitors: A new era of lipid lowering therapy.” World J. Cardiol., 26:76-91 (2017) 3. FIELDS, G.B., et al., Chapter 3 in Synthetic Peptides: A User’s Guide, ed. Grant, W.H. Freeman & Co., New York, NY, p.77 (1992) 4. HESS, C., et al., “PCSK9 Inhibitors: Mechanisms of Action, Metabolic Effects, and Clinical Outcomes.” Annul. Rev. Med., 69:17.1-17.13 (2018) 5. HOLLA, O.L., et al., “Role of the C-terminal domain of PCSK9 in degradation of the LDL receptors.” J. Lipid Res., 52(10):1787-94 (2011) 6. TRAGGIAI, E., et al., “An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus.” Nature Medicine, 10:871-875 (2004) 7. U.S. Patent No. 9,669,079, by BRUNNER, S., et al., “PCSK9 peptide combination vaccine and method of use.” (2017-06-06) 8. U.S. Patent No. 9,987,341, by CHAMPION, R., et al., “PCSK9 vaccine.” (2018-06-05) 9. WO 1990 / 014837, by VAN NEST, G., et al., “Adjuvant formulation comprising a submicron oil droplet emulsion.” (1990-12-13) SUMMARY OF THE INVENTION

[0014] The present disclosure is directed to proprotein convertase subtilisin-kexin type 9 (PCSK9) and formulations thereof for preventing and treating PCSK9-mediated disorders, including increased serum levels of low-density lipoprotein cholesterol (LDL-C) and cardiovascular (CV) events. In particular, the disclosure is directed to peptide immunogenic constructs comprising B-cell epitopes of the catalytic domain of PCSK9, compositions comprising the peptide immunogenic constructs, methods for preparing and using the peptide immunogenic constructs, and antibodies generated by the peptide immunogenic constructs.

[0015] One aspect of the present disclosure is directed to B cell epitopes of the catalytic domain of PCSK9 (residues 153-454 of SEQ ID NO: 1). The disclosed B cell epitope peptides contain about 7 to about 30 amino acids of the catalytic domain of the PCSK9 protein. In certain embodiments, the B cell epitope peptides have the amino acid sequences of SEQ ID NOs: 2-9, as shown in Table 1.

[0016] The B cell epitope peptides of the present disclosure derived from the catalytic domain of PCSK9 can be linked to heterologous helper T cell (Th) epitope peptides via an optional heterologous spacer to form peptide immunogen constructs. In certain embodiments, the heterologous spacer is any molecule or chemical structure capable of linking two amino acids and / or peptides together, and such molecules or chemical structures can include chemical compounds, naturally occurring amino acids, non-naturally occurring amino acids, or any combination thereof. The heterologous Th epitope can be any Th epitope capable of enhancing an immune response against the B cell epitope. In certain embodiments, the Th epitope is derived from a pathogen protein and has the amino acid sequence of SEQ ID NOs: 13-64 (shown in Table 2).

[0017] The peptide immunogen constructs of the present disclosure comprise a PCSK9 B cell epitope peptide covalently linked at either the N- or C-terminus to a heterologous Th epitope via an optional heterologous spacer. The peptide immunogen constructs of the present disclosure comprise a B cell epitope and a Th epitope, and have a total amino acid sequence of 20 or more. In certain embodiments, the peptide immunogen constructs have the amino acid sequences of SEQ ID NOs: 65-107 (shown in Table 3).

[0018] The PCSK9 peptide immunogen constructs of the present disclosure contain both designed B cell epitope peptides and Th epitope peptides, which work together to stimulate the production of highly specific antibodies directed against PCSK9 functional sites (including the PCSK9 and LDL-R receptor binding regions located in the catalytic domain of the PCSK9 molecule). The antibodies generated from the disclosed peptide immunogen constructs provide a therapeutic immune response to patients with PCSK9-mediated disorders, including elevated serum levels of LDL-C and CV events.

[0019] Another aspect of the present disclosure is directed to peptide compositions (including pharmaceutical compositions) comprising PCSK9 peptide immunogenic constructs. The compositions may include one or more PCSK9 peptide immunogenic constructs, a pharmaceutically acceptable delivery vehicle, an adjuvant, and / or may be formulated into an immunostimulatory complex stabilized using CpG oligomers. In certain embodiments, a mixture of PCSK9 peptide immunogenic constructs contains heterologous Th epitopes derived from different pathogens, and the use of these heterologous Th epitopes may enable coverage of a wide range of patient genetic backgrounds and increase the percentage of responders upon immunization for the prevention and / or treatment of patients with PCSK9-mediated disorders, including elevated serum levels of low-density lipoprotein cholesterol (LDL-C) and CV events.

[0020] The present disclosure also relates to antibodies against the PCSK9 peptide immunogen constructs of the present disclosure. Specifically, the PCSK9 peptide immunogen constructs of the present disclosure can stimulate the production of highly specific functional antibodies that cross-react with full-length PCSK9 protein. The antibodies of the present disclosure bind to PCSK9 with high specificity and are not often, if ever, directed against heterologous Th epitopes used for immunogenicity enhancement, which is in stark contrast to antibodies generated using traditional KLH or toxoid proteins or other biological carriers used for such peptide immunogenicity enhancement. Thus, the PCSK9 peptide immunogen constructs of the present disclosure have the ability to break immune tolerance to autologous PCSK9 and achieve a higher responder rate compared to other peptide or protein immunogens. Based on their unique characteristics and properties, the antibodies of the present disclosure induced by PCSK9 peptide immunogen constructs have the potential to provide a preventative immunotherapeutic approach to treating patients suffering from PCSK9-mediated disorders, including elevated serum levels of low-density lipoprotein cholesterol (LDL-C) and CV events.

[0021] In another aspect, the present invention provides human monoclonal antibodies against the catalytic domain of the PCSK9 molecule, which is involved in the LDL-R binding region, induced by patients administered a composition comprising a PCSK9 peptide immunogen construct of the present disclosure. A method effective for preparing human monoclonal antibodies from B cells isolated from the blood of human patients is described by Traggiai, E., et al., 2004, which is incorporated by reference.

[0022] The present disclosure is also directed to methods of preparing and using the disclosed PCSK9 peptide immunogenic constructs, compositions, and antibodies. The disclosed methods provide low-cost manufacturing and quality control of the PCSK9 peptide immunogenic constructs and compositions comprising the constructs. The disclosed methods are also directed to using the disclosed PCSK9 peptide immunogenic constructs and / or antibodies derived from the PCSK9 peptide immunogenic constructs to prevent and / or treat subjects susceptible to or suffering from PCSK9-mediated disorders, including elevated serum levels of low-density lipoprotein cholesterol (LDL-C) and CV events. The disclosed methods also include dosing regimens, dosage forms, and routes for administering the PCSK9 peptide immunogenic constructs to prevent and / or treat PCSK9-mediated disorders, including elevated serum levels of low-density lipoprotein cholesterol (LDL-C) and CV events, in subjects. [Brief explanation of the drawings]

[0023] [Figure 1] Figure 1 shows the pathway from discovery to commercialization of high-fidelity PCSK9 designer peptide immunogen constructs and their formulations for treating patients with PCSK9-mediated disorders, including elevated serum levels of low-density lipoprotein cholesterol (LDL-C) and CV events. [Figure 2] Figure 2 identifies the sequence of PCSK9 (SEQ ID NO: 1), which consists of 692 amino acid residues, of which residues 1-30 constitute the signal peptide. The remainder of the protein is generally classified into three domains: residues 31-152 constitute the prodomain, residues 153-454 constitute the catalytic domain, and residues 455-692 constitute the C-terminal (CT) domain. Binding of PCSK9 to the EGF-A repeats of the LDL-R is mediated by a patch of residues on the catalytic domain of PCSK9. The catalytic domain of PCSK9 is involved in both autocatalytic cleavage and binding of PCSK9 to the LDL-R. [Figure 3]Figure 3 identifies the amino acid residues on PCSK9 and LDL-R that bind to each other, which are regions within the PCSK9 catalytic domain around which the PCSK9 peptide immunogen constructs of the present invention are designed. [Figure 4] FIG. 4 depicts a sequence alignment of the PCSK9 catalytic domains of human (SEQ ID NO: 111), monkey (SEQ ID NO: 112), mouse (SEQ ID NO: 113), rat (SEQ ID NO: 114), and guinea pig (SEQ ID NO: 115). [Figure 5] Figure 5 illustrates PCSK9 peptide immunogen constructs (SEQ ID NOS: 65-76) designed with B cell epitopes derived from the PCSK9 catalytic domain around the site where PCSK9 and LDL-R bind to each other (SEQ ID NOS: 2-9). [Figure 6] Figure 6 illustrates immunoreactivity results with the corresponding PCSK9 B cell epitope peptides from an immunogenicity study of guinea pigs immunized with representative PCSK9 peptide immunogen constructs (SEQ ID NOS: 65-76) from the ISA51 formulation. Titers of guinea pig immune sera are expressed as Log10 titers. Immune sera were collected from 0, 3, 6, 9, and / or 12 wpi as indicated in each panel. [Figure 7] Figure 7 illustrates cross-immunoreactivity results with full-length recombinant human PCSK9 protein from an immunogenicity study of guinea pigs immunized with representative PCSK9 peptide immunogen constructs (SEQ ID NOS: 65-76) from the ISA51 formulation. Titers of guinea pig immune sera are expressed as Log10EC50. Immune sera were collected from 0, 3, 6, 9, 12, and / or 15 wpi as indicated in each panel with accompanying tables on the left. [Figure 8A]Figures 8A-8B illustrate the mean results of LDL-C levels (mg / dL) in plasma collected at each week, as shown in an immunogenicity study of guinea pigs (n=3 per group) immunized with representative PCSK9 peptide immunogen constructs from the ISA51 formulation. Figure 8A shows the results for guinea pigs immunized with peptide immunogen constructs from the N-terminal region (SEQ ID NOS: 65-67) and the central region (SEQ ID NOS: 75 and 76) of PCSK9. Figure 8B shows the results for guinea pigs immunized with peptide immunogen constructs from the C-terminal region (SEQ ID NOS: 68-74) of PCSK9. For each group of guinea pigs immunized with the corresponding PCSK9 peptide immunogen construct (SEQ ID NOS: 65-76), the percent LDL-C reduction compared to the placebo group for blood samples collected at the corresponding time point (e.g., 0, 3, 6 wpi, etc.) is shown in the accompanying table on the right. [Figure 8B] Figures 8A-8B illustrate the mean results of LDL-C levels (mg / dL) in plasma collected at each week, as shown in an immunogenicity study of guinea pigs (n=3 per group) immunized with representative PCSK9 peptide immunogen constructs from the ISA51 formulation. Figure 8A shows the results for guinea pigs immunized with peptide immunogen constructs from the N-terminal region (SEQ ID NOS: 65-67) and the central region (SEQ ID NOS: 75 and 76) of PCSK9. Figure 8B shows the results for guinea pigs immunized with peptide immunogen constructs from the C-terminal region (SEQ ID NOS: 68-74) of PCSK9. For each group of guinea pigs immunized with the corresponding PCSK9 peptide immunogen construct (SEQ ID NOS: 65-76), the percent LDL-C reduction compared to the placebo group for blood samples collected at the corresponding time point (e.g., 0, 3, 6 wpi, etc.) is shown in the accompanying table on the right. [Figure 9A]Figures 9A-9B illustrate the mean results of plasma total cholesterol (T-CHO) levels (mg / dL) collected at each week, as shown in an immunogenicity study of guinea pigs (n=3 per group) immunized with representative PCSK9 peptide immunogen constructs from the ISA51 formulation. Figure 9A shows the results for guinea pigs immunized with peptide immunogen constructs from the N-terminal region (SEQ ID NOS: 65-67) and the central region (SEQ ID NOS: 75 and 76) of PCSK9. Figure 9B shows the results for guinea pigs immunized with peptide immunogen constructs from the C-terminal region (SEQ ID NOS: 68-74) of PCSK9. For each group of guinea pigs immunized with the corresponding PCSK9 peptide immunogen construct (SEQ ID NOS: 65-76), the percent T-CHO reduction, expressed as a percentage, compared to the placebo group for blood samples collected at the corresponding time point (e.g., 0, 3, 6 wpi, etc.) is shown in the accompanying table on the right. [Figure 9B] Figures 9A-9B illustrate the mean results of plasma total cholesterol (T-CHO) levels (mg / dL) collected at each week, as shown in an immunogenicity study of guinea pigs (n=3 per group) immunized with representative PCSK9 peptide immunogen constructs from the ISA51 formulation. Figure 9A shows the results for guinea pigs immunized with peptide immunogen constructs from the N-terminal region (SEQ ID NOS: 65-67) and the central region (SEQ ID NOS: 75 and 76) of PCSK9. Figure 9B shows the results for guinea pigs immunized with peptide immunogen constructs from the C-terminal region (SEQ ID NOS: 68-74) of PCSK9. For each group of guinea pigs immunized with the corresponding PCSK9 peptide immunogen construct (SEQ ID NOS: 65-76), the percent T-CHO reduction, expressed as a percentage, compared to the placebo group for blood samples collected at the corresponding time point (e.g., 0, 3, 6 wpi, etc.) is shown in the accompanying table on the right. [Figure 10A]Figures 10A-10C illustrate results relating to the inhibition of LDL-R degradation by polyclonal antibodies from guinea pigs immunized with PCSK9 peptide immunogen constructs. Figure 10A shows the LDL assay procedure in the left panel of the figure, and the antibody dose (0, 5, 15, 100, 500, 1,000, 1,250 μg / ml)-dependent LDL uptake rate (indicating surface LDL-R expression that is not degraded by PCSK9 binding) in guinea pigs immunized with the PCSK9 peptide immunogen constructs of SEQ ID NOs: 65 and 75 as a bar graph in the right panel. [Figure 10B] Figure 10B illustrates results related to the inhibition of LDL-R degradation by polyclonal antibodies from guinea pigs immunized with PCSK9 peptide immunogen constructs. A bar graph shows antibody dose (0, 50, 250, and 500 μg / mL)-dependent LDL uptake rates from guinea pigs immunized with PCSK9 peptide immunogen constructs of SEQ ID NOs: 65, 75, 76, and 68-74. [Figure 10C] Figure 10C illustrates results related to the inhibition of LDL-R degradation by polyclonal antibodies from guinea pigs immunized with PCSK9 peptide immunogen constructs. Bar graphs show antibody dose (1,250, 1,000, 500, 100, 15, 5, and 0 μg / mL)-dependent LDL uptake rates from guinea pigs immunized with PCSK9 peptide immunogen constructs of SEQ ID NOs: 65, 75, 76, and 70. [Figure 11] Figure 11 shows immunogenicity serum titer analysis of polyclonal antibodies elicited in guinea pigs immunized with PCSK9 peptide immunogens of SEQ ID NO: 66 or 67. The graph shown in the right panel demonstrates that antibodies elicited by the peptide immunogen constructs are highly reactive to B cell epitopes of PCSK9 (SEQ ID NOs: 3 and 4) and not to Th epitopes of UBITh®1 or CpG3 oligonucleotides. [Figure 12] FIG. 12 shows the reduction in serum T-CHO and LDL levels in guinea pigs immunized with PCSK9 peptide immunogens of SEQ ID NO: 65, 66, or 67. DETAILED DESCRIPTION OF THE INVENTION

[0024] The present disclosure is directed to proprotein convertase subtilisin-kexin type 9 (PCSK9) and formulations thereof for preventing and treating PCSK9-mediated disorders, including elevated serum levels of low-density lipoprotein cholesterol (LDL-C) and CV events. In particular, the disclosure is directed to peptide immunogenic constructs comprising B-cell epitopes of the catalytic domain of PCSK9, compositions comprising the peptide immunogenic constructs, methods for preparing and using the peptide immunogenic constructs, and antibodies generated by the peptide immunogenic constructs.

[0025] One aspect of the present disclosure is directed to B cell epitopes of the catalytic domain of PCSK9 (residues 153-454 of SEQ ID NO: 1). The disclosed B cell epitope peptides contain about 7 to about 30 amino acids of the catalytic domain of the PCSK9 protein. In certain embodiments, the B cell epitope peptides have the amino acid sequences of SEQ ID NOs: 2-9, as shown in Table 1.

[0026] The B cell epitope peptides of the present disclosure derived from the catalytic domain of PCSK9 can be linked to heterologous helper T cell (Th) epitope peptides via an optional heterologous spacer to form peptide immunogen constructs. In certain embodiments, the heterologous spacer is any molecule or chemical structure capable of linking two amino acids and / or peptides together, and such molecules or chemical structures can include chemical compounds, naturally occurring amino acids, non-naturally occurring amino acids, or any combination thereof. The heterologous Th epitope can be any Th epitope capable of enhancing an immune response against the B cell epitope. In certain embodiments, the Th epitope is derived from a pathogen protein and has the amino acid sequence of SEQ ID NOs: 13-64 (shown in Table 2).

[0027] In certain embodiments, heterologous Th epitopes used to enhance PCSK9 B cell epitope peptides are derived from natural pathogens (EBV BPLF1 (SEQ ID NO:51), EBV CP (SEQ ID NO:48), Clostridium tetani (SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:43, SEQ ID NOs:45-47), cholera toxin (SEQ ID NO:20), and Schistosoma mansoni (SEQ ID NO:19)), as well as idealized artificial Th epitopes derived from measles virus fusion proteins (MVF1-5) and hepatitis B surface antigens (HBsAg1-3), and these heterologous Th epitopes are in the form of either single sequences or combination sequences (e.g., SEQ ID NO:14, SEQ ID NOs:21-38, and SEQ ID NOs:53-64).

[0028] The peptide immunogen constructs of the present disclosure comprise a PCSK9 B cell epitope peptide covalently linked at either the N- or C-terminus to a heterologous Th epitope via an optional heterologous spacer. The peptide immunogen constructs of the present disclosure comprise a PCSK9 B cell epitope and a Th epitope, and have a total amino acid sequence of 20 or more. In certain embodiments, the peptide immunogen constructs have the amino acid sequences of SEQ ID NOs: 65-107 (shown in Table 3).

[0029] The PCSK9 peptide immunogen constructs of the present disclosure comprise both designed B cell epitope peptides and Th epitope peptides, which work together to stimulate the production of highly specific antibodies directed against PCSK9 functional sites (including the PCSK9 and LDL-R receptor binding regions located in the catalytic domain of the PCSK9 molecule) and provide a therapeutic immune response in patients with PCSK9-mediated disorders, including elevated serum levels of low-density lipoprotein cholesterol (LDL-C) and CV events.

[0030] Another aspect of the present disclosure is directed to peptide compositions comprising PCSK9 peptide immunogen constructs. In some embodiments, the composition contains one peptide immunogen construct. In other embodiments, the peptide composition comprises a mixture of PCSK9 peptide immunogen constructs. In certain embodiments, the mixture of PCSK9 peptide immunogen constructs comprises heterologous Th epitopes derived from different pathogens, and the use of these heterologous Th epitopes may enable coverage of a wide range of patient genetic backgrounds and increase the percentage of responders upon immunization for the prevention and / or treatment of patients with PCSK9-mediated disorders, including elevated serum levels of low-density lipoprotein cholesterol (LDL-C) and CV events.

[0031] Synergistic enhancement with PCSK9 immunogen constructs can be observed with the peptide compositions of the present disclosure. The antibody responses obtained by administering such PCSK9 peptide immunogen construct-containing compositions were largely (>90%) focused on the desired cross-reactivity against the PCSK9 site(s) or LDL-R receptor binding region peptides (SEQ ID NOS: 2-9), with little, if any, directed against the heterologous Th epitope used for immunogenic enhancement. The immune response using the disclosed Th epitope-containing peptide immunogen constructs stands in stark contrast to standard methods using conventional carrier proteins (such as KLH, toxoids, or other biological carriers) for such peptide antigenic enhancement.

[0032] The present disclosure is also directed to pharmaceutical compositions and formulations thereof for preventing and / or treating patients with PCSK9-mediated disorders, including elevated serum levels of low-density lipoprotein cholesterol (LDL-C) and CV events. In some embodiments, the pharmaceutical compositions are formulated into stabilized immunostimulatory complexes formed through electrostatic association by mixing a peptide composition comprising a PCSK9 peptide immunogen construct, or a mixture of constructs, with a CpG oligomer. Such stabilized immunostimulatory complexes further enhance PCSK9 peptide immunogenicity to achieve desired cross-reactivity with full-length PCSK9 protein.

[0033] In other embodiments, pharmaceutical compositions comprising the PCSK9 peptide immunogen constructs of the present disclosure or pharmaceutical compositions comprising a mixture of constructs are formulated with a pharmaceutically acceptable delivery vehicle or adjuvant, such as an inorganic salt (including alumgel (ALHYDROGEL) or aluminum phosphate (ADJU-PHOS)), to form a suspension, or with MONTANIDE™ ISA51 or MONTANIDE™ ISA720 as an adjuvant to form a water-in-oil emulsion, which suspension or water-in-oil emulsion may be used to prevent and / or treat patients with PCSK9-mediated disorders, including elevated serum levels of low-density lipoprotein cholesterol (LDL-C) and CV events.

[0034] The present disclosure also relates to antibodies against the PCSK9 peptide immunogen constructs of the present disclosure. Specifically, the PCSK9 peptide immunogen constructs of the present disclosure can stimulate the production of highly specific functional antibodies that cross-react with full-length PCSK9 protein. The antibodies of the present disclosure bind to PCSK9 with high specificity and are not often, if ever, directed against heterologous Th epitopes used for immunogenicity enhancement, which is in stark contrast to antibodies generated using traditional KLH or toxoid proteins or other biological carriers used for such peptide immunogenicity enhancement. Thus, the PCSK9 peptide immunogen constructs of the present disclosure have the ability to break immune tolerance to autologous PCSK9 and achieve a higher responder rate compared to other peptide or protein immunogens.

[0035] In some embodiments, the disclosed antibodies target and specifically bind to PCSK9 and LDL-R receptor binding sites (e.g., SEQ ID NOS: 2-9) on the catalytic domain of the PCSK9 molecule when the peptide immunogen constructs are administered to a subject. The highly specific antibodies elicited by these PCSK9 peptide immunogen constructs can inhibit PCSK9 and LDL-R receptor binding and downstream cellular events, including internalization and cellular processing of the PCSK9 and LDL-R complex, which leads to LDL-R degradation, resulting in effective prevention and / or treatment of patients with PCSK9-mediated disorders, including elevated serum levels of low-density lipoprotein cholesterol (LDL-C) and CV events.

[0036] Based on their unique characteristics and properties, the antibodies of the present disclosure induced by PCSK9 peptide immunogen constructs have the potential to provide a preventative immunotherapeutic approach to treating patients suffering from PCSK9-mediated disorders, including elevated serum levels of low-density lipoprotein cholesterol (LDL-C) and CV events.

[0037] In another aspect, the present invention provides human monoclonal antibodies against the catalytic domain of the PCSK9 molecule, which is involved in the LDL-R binding region, induced by patients administered a composition comprising a PCSK9 peptide immunogen construct of the present disclosure. A method effective for preparing human monoclonal antibodies from B cells isolated from the blood of human patients is described by Traggiai, E., et al., 2004, which is incorporated by reference.

[0038] The present disclosure is also directed to methods for preparing the disclosed PCSK9 peptide immunogenic constructs, compositions, and antibodies. The disclosed methods provide low-cost manufacturing and quality control of PCSK9 peptide immunogenic constructs and compositions comprising the constructs, which can be used in methods for treating patients suffering from PCSK9-mediated disorders, including elevated serum levels of low-density lipoprotein cholesterol (LDL-C) and CV events.

[0039] The present disclosure also includes methods for preventing and / or treating subjects susceptible to or suffering from PCSK9-mediated disorders (including elevated serum levels of low-density lipoprotein cholesterol (LDL-C) and CV events) using the PCSK9 peptide immunogenic constructs of the present disclosure and / or antibodies derived from the PCSK9 peptide immunogenic constructs. Methods for preventing and / or treating patients with PCSK9-mediated disorders include administering to the subject a composition comprising a PCSK9 peptide immunogenic construct or a mixture of constructs of the present disclosure. In certain embodiments, the composition utilized in the method comprises a PCSK9 peptide immunogenic construct of the present disclosure in the form of a stable immunostimulatory complex (to which an adjuvant may further be added) with a negatively charged oligonucleotide (such as a CpG oligomer) formed via electrostatic association.

[0040] The methods of the disclosure also include dosing regimens, dosage forms, and routes for administering PCSK9 peptide immunogen constructs to prevent and / or treat patients with PCSK9-mediated disorders of interest, including elevated serum levels of low-density lipoprotein cholesterol (LDL-C) and CV events.

[0041] overview The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All references or portions of references cited in this application are expressly incorporated herein by reference in their entirety for all purposes.

[0042] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. Thus, the phrase "comprising A or B" means including A or B, or A and B. It will be further understood that all amino acid sizes and all molecular weight or molecular mass values ​​given for polypeptides are approximate and are provided for illustrative purposes. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terminology, will control. Furthermore, the materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0043] PCSK9 peptide immunogen construct The present disclosure provides peptide immunogen constructs comprising a B cell epitope peptide having about 7 to about 30 amino acids having the amino acid sequence of PCSK9 (SEQ ID NO: 1). In certain embodiments, the B cell epitope peptide has an amino acid sequence selected from SEQ ID NOs: 2 to 9 (shown in Table 1), which are located in the N-terminal, central, and C-terminal regions of the catalytic domain of PCSK9. In some embodiments, the B cell epitope peptide has an amino acid sequence from PCSK9 and the LDL-R receptor binding region (e.g., SEQ ID NOs: 2 to 6 and 8 to 9 shown in Table 1).

[0044] The B cell epitope can be covalently linked directly or via an optional heterologous spacer to a heterologous helper T cell (Th) epitope derived from a pathogen protein (e.g., SEQ ID NOS: 13-64 as shown in Table 2). Such constructs contain both engineered B cell and Th epitopes that work together to stimulate the generation of highly specific antibodies that cross-react with full-length human PCSK9 (SEQ ID NO: 1).

[0045] As used herein, the phrase "PCSK9 peptide immunogen construct" or "peptide immunogen construct" refers to a peptide comprising 20 or more amino acids, including (a) a B cell epitope having more than about 7 contiguous amino acid residues derived from the full-length PCSK9 protein (SEQ ID NO: 1), (b) a heterologous Th epitope, and (c) an optional heterologous spacer.

[0046] In certain embodiments, the PCSK9 peptide immunogen construct has the following formula: (Th) m -(A) n -(PCSK9 functional B cell epitope peptide)-X or (PCSK9 functional B cell epitope peptide)-(A) n -(Th) m -X or (Th) m-(A) n -(PCSK9 functional B cell epitope peptide)-(A) n -(Th) m -X It can be expressed as During the ceremony, Th is a heterologous helper T cell epitope; A is a heterologous spacer, (PCSK9 functional B cell epitope peptide) is a B cell epitope peptide having 7 to 30 PCSK9-derived amino acid residues involved in either receptor binding or receptor activation, X is the α-COOH or α-CONH2 of an amino acid, m is from 1 to about 4; n is 0 to about 10.

[0047] The PCSK9 peptide immunogen constructs of the present disclosure were designed and selected based on a number of rationales, including the following: i.PCSK9 B cell epitope peptides are non-immunogenic by themselves, thereby avoiding the activation of autologous T cells, ii. The PCSK9 B cell epitope peptide can be made immunogenic by using a protein carrier or a strong helper T cell epitope(s); iii. When the PCSK9 B cell epitope peptide is made immunogenic and administered to a host, the host a. Induces high titer antibodies selectively directed against PCSK9 B cell epitope(s) and not against protein carrier or helper T cell epitope(s); b. Breaking immune tolerance and generating highly specific antibodies that are cross-reactive with the PCSK9 protein (SEQ ID NO: 1); c. Generating highly specific antibodies that can inhibit PCSK9 and LDL-R receptor binding along with the associated downstream cellular events that lead to LDL-R degradation, thereby causing increased LDL-C uptake by LDL-R expressing cells; d. The levels of LDL-C and T-CHO will be reduced in the subject's plasma.

[0048] The PCSK9 peptide immunogen constructs and formulations thereof of the present disclosure can effectively function as pharmaceutical compositions to prevent and / or treat subjects susceptible to or suffering from PCSK9-mediated disorders, including elevated serum levels of low-density lipoprotein cholesterol (LDL-C) and CV events in the subject.

[0049] The various components of the PCSK9 peptide immunogen constructs of the present disclosure are described in further detail below.

[0050] a. PCSK9-derived B cell epitope peptide The present disclosure is directed to proprotein convertase subtilisin-kexin type 9 (PCSK9) and formulations thereof for preventing and treating PCSK9-mediated disorders, including elevated serum levels of low-density lipoprotein cholesterol (LDL-C) and CV events. The present disclosure also is directed to peptide immunogenic constructs comprising B-cell epitopes of the catalytic domain of PCSK9, compositions comprising the peptide immunogenic constructs, methods for preparing and using the peptide immunogenic constructs, and antibodies generated by the peptide immunogenic constructs.

[0051] The present disclosure is directed to novel peptide compositions for generating high-titer antibodies specific for proprotein convertase subtilisin kexin type 9 (PCSK9) (e.g., SEQ ID NO: 1). The catalytic domain of PCSK9 (SEQ ID NO: 111) is used as a B-cell epitope target. The generation of antibodies directed to irrelevant sites on other regions of PCSK9 or on carrier proteins is minimized by the site specificity of the peptide immunogen constructs, thereby providing a high safety margin.

[0052] The PCSK9 gene is located on human chromosome 1p32 and encodes a 692-amino acid serine protease. PCSK9 (GenBank accession number: EAX06660) has the amino acid sequence of SEQ ID NO: 1, as shown in both Table 1 and Figure 2. The PCSK9 protein consists of a signal peptide (residues 1-30), a prodomain (residues 31-152), a catalytic domain (residues 153-454, SEQ ID NO: 111), and a C-terminal (CT) domain (455-692) consisting of three modules: CM1 (residues 457-527), CM2 (residues 534-601), and CM3 (residues 608-692). Binding of PCSK9 to the EGF-A repeats of the LDL-R is mediated by a patch of residues on the catalytic domain of PCSK9. Figure 3 identifies the amino acid residues on PCSK9 and the LDL-R that bind to each other. The catalytic domain of PCSK9 is involved in both autocatalytic cleavage and binding of PCSK9 to LDL-R.

[0053] One aspect of the present disclosure is to prevent and / or treat PCSK9-mediated disorders, including elevated serum levels of low-density lipoprotein cholesterol (LDL-C) and CV events in a subject. Accordingly, the present disclosure is directed to peptide immunogen constructs targeting the catalytic domain (SEQ ID NO: 111) of the full-length PCSK9 protein (SEQ ID NO: 1), as well as formulations thereof, for preventing and / or treating PCSK9-mediated disorders.

[0054] The B cell epitope portion of the PCSK9 peptide immunogen construct may comprise about 7 to about 30 amino acids derived from the catalytic domain (SEQ ID NO: 111) of the PCSK9 protein represented by SEQ ID NO: 1. In certain embodiments, the B cell epitope peptides are screened and selected based on design rationale and contain the amino acid sequences of SEQ ID NOs: 2 to 9 shown in Table 1.

[0055] The PCSK9 B-cell epitope peptides of the present disclosure also include amino acid sequences that are immunologically functional analogs or homologs of PCSK9. Functional immunological analogs or immunological homologs of PCSK9 B-cell epitope peptides include variants that retain substantially the same immunogenicity as the original peptide. Immunologically functional analogs may have conservative substitutions at certain amino acid positions, alterations in overall charge, covalent attachment to another moiety, or additions, insertions, or deletions of amino acids, and / or any combination thereof. Examples of immunologically functional analogs are shown in Table 1 (e.g., SEQ ID NO:2 vs. SEQ ID NO:8, SEQ ID NO:5 vs. SEQ ID NO:9, and SEQ ID NOs:2, 3, and 4).

[0056] Antibodies generated from peptide immunogen constructs containing the disclosed B-cell epitopes from PCSK9 are highly specific for and cross-reactive with full-length human PCSK9 (SEQ ID NO: 1).

[0057] b. Heterologous helper T cell epitopes (Th epitopes) The present disclosure provides peptide immunogen constructs comprising a B-cell epitope from PCSK9 covalently linked either directly or via an optional heterologous spacer to a heterologous helper T-cell (Th) epitope.

[0058] The heterologous Th epitope in the peptide immunogen construct enhances the immunogenicity of the PCSK9 B cell epitope moiety, thereby facilitating the generation of specific, high-titer antibodies directed against the optimized target PCSK9 B cell epitope peptides screened and selected based on the design rationale.

[0059] The term "heterologous" as used herein refers to an amino acid sequence that is not part of the wild-type sequence of PCSK9, or an amino acid sequence that is not homologous to the wild-type sequence of PCSK9. Thus, a heterologous Th epitope is a Th epitope that is derived from an amino acid sequence that is not naturally found in PCSK9 (i.e., the Th epitope is not derived from PCSK9). Because such a Th epitope is heterologous to PCSK9, covalent linkage of such a heterologous Th epitope to the PCSK9 B-cell epitope peptide does not extend the native amino acid sequence of PCSK9 toward either the N-terminus or the C-terminus.

[0060] The heterologous Th epitope of the present disclosure can be any Th epitope that does not have an amino acid sequence naturally found in PCSK9. The Th epitope can also have motifs that non-selectively bind to MHC class II molecules of multiple species. In certain embodiments, the Th epitope contains multiple motifs that non-selectively bind to MHC class II molecules, allowing for maximal activation of helper T cells, thereby initiating and controlling the immune response. The Th epitope is preferably immunologically static by itself, i.e., very few, if any, antibodies generated by the PCSK9 peptide immunogen construct will be directed against the Th epitope, thereby allowing for the generation of a highly focused immune response directed against the target B-cell epitope peptide of the PCSK9 molecule.

[0061] Th epitopes of the present disclosure include, but are not limited to, amino acid sequences derived from foreign pathogens (e.g., those exemplified in Table 2 (e.g., SEQ ID NOS: 13-64)). Additionally, heterologous Th epitopes include idealized artificial Th epitopes (e.g., SEQ ID NOS: 14, 21, 25-29, 31-32, 34-35, 37-38, 53-56, 58-59, and 61-64) and idealized combinatorial artificial Th epitopes (e.g., SEQ ID NOS: 24, 30, 33, 36, 57, and 60). A combination of idealized artificial Th epitopes includes a mixture of amino acid residues designated at specific positions within a peptide framework based on the variable residues of homologs to that particular peptide. A collection of combinatorial peptides can be synthesized in a single process by adding a designated mixture of protected amino acids to specific positions in place of one specific amino acid during the synthesis process. Such a collection of combined heterologous Th epitope peptides may enable broader Th epitope coverage for animals with diverse genetic backgrounds. Representative combined sequences of heterologous Th epitope peptides include SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 57, and SEQ ID NO: 60 shown in Table 2. The epitope peptides of the present invention confer broad reactivity and immunogenicity to animals and patients from genetically diverse populations.

[0062] c. heterologous spacer The PCSK9 peptide immunogen constructs of the present disclosure optionally include a heterologous spacer covalently linking the PCSK9 B-cell epitope peptide to the heterologous helper T-cell (Th) epitope.

[0063] As noted above, the term "heterologous" refers to an amino acid sequence that is derived from an amino acid sequence that is not part of the native sequence of PCSK9, or that is not homologous to the native sequence of PCSK9. Thus, even if a heterologous spacer is covalently linked to the PCSK9 B-cell epitope peptide, the native amino acid sequence of PCSK9 is not extended in either the N- or C-terminal direction because the spacer is heterologous to the PCSK9 sequence.

[0064] A spacer is any molecule or chemical structure capable of linking two amino acids and / or peptides together. The length or polarity of the spacer can vary depending on the application. Attachment of the spacer can be via an amide or carboxyl bond, although other functionalities can also be utilized. Spacers can include chemical compounds, naturally occurring amino acids, or non-naturally occurring amino acids.

[0065] A spacer can impart structural features to a PCSK9 peptide immunogen construct. Structurally, the spacer physically separates the Th epitope from the B cell epitope of the PCSK9 fragment. The physical separation by the spacer can disrupt any artificial secondary structure created by linking the Th epitope with the B cell epitope. Furthermore, physically separating the epitopes by the spacer can eliminate interference between the Th cell response and / or the B cell response. Furthermore, the spacer can be designed to create or modify the secondary structure of the peptide immunogen construct. For example, the spacer can be designed to act as a flexible hinge to enhance the separation of the Th epitope and the B cell epitope. A flexible hinge spacer can also improve the efficiency of the interaction between the presented peptide immunogen and appropriate Th cells and B cells, thereby enhancing the immune response to the Th epitope and the B cell epitope. Examples of sequences encoding flexible hinges are those found in immunoglobulin heavy chain hinge regions, which are often rich in proline. One particularly useful flexible hinge that can be used as a spacer is provided by the sequence Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO: 10), where Xaa is any amino acid, preferably aspartic acid.

[0066] The spacer can also confer functional characteristics to the PCSK9 peptide immunogen construct. For example, the spacer can be designed to alter the overall charge of the PCSK9 peptide immunogen construct, thereby affecting the solubility of the peptide immunogen construct. Furthermore, altering the overall charge of the PCSK9 peptide immunogen construct can affect the ability of the peptide immunogen construct to bind to other compounds and reagents. As discussed in more detail below, the PCSK9 peptide immunogen construct can form stable immunostimulatory complexes with highly charged oligonucleotides (e.g., CpG oligomers) through electrostatic association. The overall charge of the PCSK9 peptide immunogen construct is important for the formation of such stable immunostimulatory complexes.

[0067] Compounds that can be used as spacers include, but are not limited to, (2-aminoethoxy)acetic acid (AEA), 5-aminovaleric acid (AVA), 6-aminocaproic acid (Ahx), 8-amino-3,6-dioxaoctanoic acid (AEEA, miniPEG1), 12-amino-4,7,10-trioxadodecanoic acid (miniPEG2), 15-amino-4,7,10,13-tetraoxapentadecanoic acid (miniPEG3), trioxatridecane-succinic acid (Ttds), 12-amino-dodecanoic acid, Fmoc-5-amino-3-oxapentanoic acid (O1Pen), and the like.

[0068] Naturally occurring amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0069] Non-naturally occurring amino acids include, but are not limited to, ε-N-lysine, β-alanine, ornithine, norleucine, norvaline, hydroxyproline, thyroxine, γ-aminobutyric acid, homoserine, citrulline, aminobenzoic acid, 6-aminocaproic acid (Aca; 6-aminohexanoic acid), hydroxyproline, mercaptopropionic acid (MPA), 3-nitro-tyrosine, pyroglutamic acid, and the like.

[0070] The spacer included in the PCSK9 peptide immunogen construct can be covalently linked to either the N- or C-terminus of the Th epitope and the PCSK9 B cell epitope peptide. In some embodiments, the spacer is covalently linked to the C-terminus of the Th epitope and the N-terminus of the PCSK9 B cell epitope peptide. In other embodiments, the spacer is covalently linked to the C-terminus of the PCSK9 B cell epitope peptide and the N-terminus of the Th epitope. In certain embodiments, multiple spacers can be used, for example, when multiple Th epitopes are present in the PCSK9 peptide immunogen construct. When multiple spacers are used, each spacer can be the same as or different from each other. Furthermore, when multiple Th epitopes are present in the PCSK9 peptide immunogen construct, the Th epitopes can be separated by a spacer, which can be the same as or different from the spacer used to separate the Th epitope from the PCSK9 B cell epitope peptide. There are no restrictions on the placement of the spacer in relation to the Th epitope or PCSK9 B cell epitope peptide.

[0071] In certain embodiments, the heterologous spacer is a naturally occurring amino acid or a non-naturally occurring amino acid. In other embodiments, the spacer comprises a plurality of naturally occurring amino acids or non-naturally occurring amino acids. In certain embodiments, the spacer is Lys-, Gly-, Lys-Lys-Lys-, (α,ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 11), or Lys-Lys-Lys-ε-N-Lys (SEQ ID NO: 12).

[0072] d. Specific Embodiments of PCSK9 Peptide Immunogen Constructs In certain embodiments, the PCSK9 peptide immunogen construct has the following formula: (Th) m -(A) n -(PCSK9 functional B cell epitope peptide)-X or (PCSK9 functional B cell epitope peptide)-(A) n -(Th) m -X or (Th) m -(A) n -(PCSK9 functional B cell epitope peptide)-(A) n -(Th) m -X It can be expressed as During the ceremony, Th is a heterologous helper T cell epitope; A is a heterologous spacer, (PCSK9 functional B cell epitope peptide) is a B cell epitope peptide having 7 to 30 amino acid residues derived from the catalytic domain of PCSK9, which is involved in PCSK9 and LDL-R receptor binding, X is the α-COOH or α-CONH2 of an amino acid, m is from 1 to about 4; n is 0 to about 10.

[0073] The B cell epitope peptide may comprise about 7 to about 30 amino acids derived from the catalytic domain of the full-length PCSK9 protein represented by SEQ ID NO: 1 (SEQ ID NO: 111). In certain embodiments, the B cell epitope peptide has an amino acid sequence selected from SEQ ID NOs: 2 to 9 (shown in Table 1), which are located in the N-terminal, central, and C-terminal regions of the catalytic domain of PCSK9. In some embodiments, the B cell epitope peptide has an amino acid sequence from PCSK9 and the LDL-R receptor binding region (e.g., SEQ ID NOs: 2 to 6 and 8 to 9 shown in Table 1).

[0074] The heterologous Th epitope in the PCSK9 peptide immunogen construct has an amino acid sequence selected from any of SEQ ID NOS: 13-64, and combinations thereof, as shown in Table 2. In some embodiments, multiple Th epitopes are present in the PCSK9 peptide immunogen construct.

[0075] The optional heterologous spacer is selected from Lys-, Gly-, Lys-Lys-Lys-, (α,ε-N)Lys, Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO: 10), ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 11), Lys-Lys-Lys-ε-N-Lys (SEQ ID NO: 12), and any combination thereof, where Xaa is any amino acid but is preferably aspartic acid. In certain embodiments, the heterologous spacer is ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 11) or Lys-Lys-Lys-ε-N-Lys (SEQ ID NO: 12).

[0076] In certain embodiments, the PCSK9 peptide immunogenic construct has an amino acid sequence selected from any of SEQ ID NOs: 65-107 shown in Table 3.

[0077] The Th epitopes that make up the PCSK9 peptide immunogen constructs are simultaneously generated in tandem with PCSK9 fragments in a single solid-phase peptide synthesis. Th epitopes also include immunological analogs of Th epitopes. Immunological Th analogs include immunopotentiating analogs, cross-reactive analogs, and segments of any of these Th epitopes that are sufficient to enhance or stimulate an immune response to PCSK9 B-cell epitope peptides.

[0078] The Th epitope contained in the PCSK9 peptide immunogen construct can be covalently linked to either the N-terminus or C-terminus of the PCSK9 B cell epitope peptide.In some embodiments, the Th epitope is covalently linked to the N-terminus of the PCSK9 B cell epitope peptide.In other embodiments, the Th epitope is covalently linked to the C-terminus of the PCSK9 B cell epitope peptide.In certain embodiments, multiple Th epitopes are covalently linked to the PCSK9 B cell epitope peptide.When multiple Th epitopes are linked to the PCSK9 B cell epitope peptide, each Th epitope can have the same amino acid sequence or different amino acid sequences.Furthermore, when multiple Th epitopes are linked to the PCSK9 B cell epitope peptide, these Th epitopes can be arranged in any order. For example, these Th epitopes can be contiguously linked to the N-terminus of the PCSK9 B cell epitope peptide, or contiguously linked to the C-terminus of the PCSK9 B cell epitope peptide. Alternatively, a Th epitope can be covalently linked to the N-terminus of the PCSK9 B cell epitope peptide, and another Th epitope can be covalently linked to the C-terminus of the PCSK9 B cell epitope peptide. There is no limitation on the arrangement of the Th epitope in relation to the PCSK9 B cell epitope peptide.

[0079] In some embodiments, the Th epitope is covalently linked directly to the PCSK9 B-cell epitope peptide, hi other embodiments, the Th epitope is covalently linked to the PCSK9 fragment via a heterologous spacer.

[0080] e. Variants, homologues, and functional analogues Variants and analogs of the above immunogenic peptide constructs that induce and / or cross-react with antibodies against preferred PCSK9 B-cell epitope peptides can also be used. Analogs (including allelic, species, and induced variants) typically differ from the naturally occurring peptide at one, two, or a few positions, often by conservative substitutions. Analogs typically have at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the naturally occurring peptide. Some analogs may also contain unnatural amino acids or N- or C-terminal amino acid modifications at one, two, or a few positions.

[0081] Variants that are functional analogs can have conservative substitutions at certain amino acid positions, altered overall charge, covalent attachment to another moiety, or additions, insertions, or deletions of amino acids, and / or any combination thereof.

[0082] Conservative substitutions are those in which one amino acid residue is replaced with another amino acid residue having similar chemical properties. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0083] In certain embodiments, a functional analog is at least 50% identical to the original amino acid sequence. In other embodiments, a functional analog is at least 80% identical to the original amino acid sequence. In yet other embodiments, a functional analog is at least 85% identical to the original amino acid sequence. In yet other embodiments, a functional analog is at least 90% identical to the original amino acid sequence.

[0084] Functional immunological analogs of the Th epitope peptides are also useful and are included as part of the present invention. Functional immunological Th analogs can include conservative substitutions, additions, deletions, and insertions of one to about five amino acid residues in the Th epitope that do not essentially alter the Th stimulatory function of the Th epitope. Conservative substitutions, additions, and insertions can be achieved using natural or unnatural amino acids, as described above for the PCSK9 B cell epitope peptides. Table 2 shows additional variations of functional analogs of the Th epitope peptides. Specifically, SEQ ID NO:14 and SEQ ID NO:21 of MvF1 Th and MvF2 Th are functional analogs of SEQ ID NOs:31-33 and SEQ ID NO:37 of MvF4 and MvF5, respectively, in that the amino acid frames of these sequences differ by the deletion of two amino acids from each of the N- and C-termini (SEQ ID NO:14 and SEQ ID NO:21) or the inclusion of two amino acids at each of the N- and C-termini (SEQ ID NOs:31-33 and SEQ ID NO:37). The differences between these two sets of similar sequences are not expected to affect the function of the Th epitopes contained within these sequences. Thus, functional immunological Th analogs include several versions of Th epitopes derived from measles virus fusion protein (MvF1-4 Th) (SEQ ID NO: 14, SEQ ID NO: 21, SEQ ID NO: 22-24, SEQ ID NO: 31-33, SEQ ID NO: 53-57, and SEQ ID NO: 58-60), as well as several versions of Th epitopes derived from hepatitis surface protein (HBsAg1-3 Th) (SEQ ID NO: 25-30, SEQ ID NO: 34-36, SEQ ID NO: 38, and SEQ ID NO: 62-64).

[0085] composition The present disclosure also provides compositions comprising the PCSK9 immunogenic peptide constructs of the present disclosure.

[0086] a. Peptide composition Compositions comprising the PCSK9 peptide immunogenic constructs of the present disclosure can be in liquid or solid / lyophilized form. Liquid compositions can contain water, buffers, solvents, salts, and / or any other acceptable reagents that do not alter the structural or functional properties of the PCSK9 peptide immunogenic constructs. Peptide compositions can include one or more of the PCSK9 peptide immunogenic constructs of the present disclosure.

[0087] b. Pharmaceutical Compositions The present disclosure is also directed to pharmaceutical compositions comprising the PCSK9 peptide immunogenic constructs of the present disclosure.

[0088] The pharmaceutical composition may comprise a carrier and / or other additives in a pharmaceutically acceptable delivery system. Thus, the pharmaceutical composition may comprise a pharmaceutically effective amount of a PCSK9 peptide immunogen construct together with a pharmaceutically acceptable carrier, adjuvant, and / or other pharmaceutical additives (such as diluents, additives, stabilizers, preservatives, solubilizers, buffers, and the like).

[0089] The pharmaceutical composition may include one or more adjuvants, which act to promote, prolong, or enhance the immune response to the PCSK9 peptide immunogen construct without themselves having any particular antigenic activity. Adjuvants used in the pharmaceutical composition may include oils, oil emulsions, aluminum salts, calcium salts, immunostimulating complexes, bacterial and viral agents, virosomes, carbohydrates, cytokines, and polymeric microparticles. In certain embodiments, the adjuvant is alum (potassium aluminum phosphate), aluminum phosphate (e.g., ADJU-PHOS®), aluminum hydroxide (e.g., ALHYDROGEL®), calcium phosphate, incomplete Freund's adjuvant (IFA), complete Freund's adjuvant, MF59, adjuvant 65, Lipovant, ISCOM, liposyn, saponin, squalene, L121, EMULSIGEN®, monophosphoryl lipid A (MPL), QuilA, QS21, The adjuvant may be selected from MONTANIDE® ISA35, ISA50V, ISA50V2, ISA51, ISA206, ISA720, liposomes, phospholipids, peptidoglycan, lipopolysaccharide (LPS), ASO1, ASO2, ASO3, ASO4, AF03, lipophilic phospholipid (lipid A), gamma inulin, algammulin, glucan, dextran, glucomannan, galactomannan, levan, xylan, dimethyldioctadecylammonium bromide (DDA), and other adjuvants and emulsifiers.

[0090] In some embodiments, the pharmaceutical composition comprises MONTANIDE™ ISA51 (an oil adjuvant composition composed of vegetable oil and mannide oleate for the production of a water-in-oil emulsion), TWEEN® 80 (also known as polysorbate 80 or polyoxyethylene(20) sorbitan oleate monoester), a CpG oligonucleotide, and / or any combination thereof. In other embodiments, the pharmaceutical composition is a water-in-oil-in-water (i.e., w / o / w) emulsion comprising EmulsIL-6n or EmulsIL-6n D as an adjuvant.

[0091] The pharmaceutical composition may also include pharmaceutically acceptable additives or excipients, for example, antioxidants, binders, buffers, fillers, carriers, chelating agents, colorants, diluents, disintegrants, emulsifiers, excipients, gelling agents, pH buffers, preservatives, solubilizers, stabilizers, and the like.

[0092] The pharmaceutical compositions can be formulated as immediate-release or sustained-release formulations. Furthermore, the pharmaceutical compositions can be formulated to induce systemic or local mucosal immunity through encapsulation and co-administration of immunogens with microparticles. Such delivery systems are readily determined by those skilled in the art.

[0093] The pharmaceutical composition can be prepared as an injection (as a solution or suspension). The liquid medium containing the PCSK9 peptide immunogen construct can also be prepared before injection. The pharmaceutical composition can be administered by any suitable application mode (e.g., intradermal, intravenous, intraperitoneal, intramuscular, intranasal, oral, subcutaneous, etc.) in any suitable delivery device. In certain embodiments, the pharmaceutical composition is formulated for intravenous administration, subcutaneous administration, intradermal administration, or intramuscular administration. Pharmaceutical compositions suitable for other administration modes, including oral and intranasal application, can also be prepared.

[0094] The pharmaceutical composition may also be formulated into a suitable unit dosage form. In some embodiments, the pharmaceutical composition comprises about 0.1 μg to about 1 mg of the PCSK9 peptide immunogen construct per kg of body weight. The effective dose of the pharmaceutical composition may vary depending on many different factors, including the means of administration, the target site, the patient's physiological condition, whether the patient is human or animal, other administered drugs, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, although non-human mammals, including transgenic mammals, may also be treated. If the pharmaceutical composition is to be delivered in multiple doses, it may be conveniently divided into appropriate amounts per unit dosage form. The dosage will depend on the age, weight, and overall health of the subject, as is well known in the therapeutic field.

[0095] In some embodiments, the pharmaceutical composition comprises multiple PCSK9 peptide immunogen constructs. A pharmaceutical composition comprising a mixture of multiple PCSK9 peptide immunogen constructs can synergistically enhance the immune efficacy of the constructs. A pharmaceutical composition comprising multiple PCSK9 peptide immunogen constructs can improve the immune response to the PCSK9 peptide immunogen constructs because broader MHC class II coverage can extend the reach of the genetic population.

[0096] In some embodiments, the pharmaceutical composition comprises a PCSK9 peptide immunogenic construct selected from SEQ ID NOs: 65-107 (Table 3), and homologs, analogs, and / or combinations thereof.

[0097] In certain embodiments, PCSK9 peptide immunogen constructs (SEQ ID NOS: 91-93) containing heterologous Th epitopes derived from MVF and HBsAg in combination (SEQ ID NOS: 24, 30, and 36, respectively) were mixed in an equimolar ratio and used in formulations to maximize coverage of host populations with diverse genetic backgrounds. Furthermore, the antibody responses elicited by PCSK9 peptide immunogen constructs (e.g., those utilizing UBITh®1 (SEQ ID NOS: 65)) were largely (>90%) focused on the desired cross-reactivity against the PCSK9 B-cell epitope peptide, with little, if any, directed against the heterologous Th epitope used to enhance immunogenicity.

[0098] This is in stark contrast to conventional proteins (such as KLH) or other biological protein carriers used to enhance the immunogenicity of such PCSK9 peptides.

[0099] In other embodiments, pharmaceutical compositions comprising a peptide composition (e.g., comprising a mixture of PCSK9 peptide immunogen constructs) in contact with an inorganic salt (including alumgel or aluminum phosphate) as an adjuvant to form a suspension formulation were used for administration to a host.

[0100] Pharmaceutical compositions comprising PCSK9 peptide immunogenic constructs can be used to elicit an immune response in the host upon administration, leading to the production of antibodies.

[0101] c. Immunostimulatory complex The present disclosure also relates to pharmaceutical compositions comprising a PCSK9 peptide immunogen construct in the form of an immunostimulatory complex with a CpG oligonucleotide. Such immunostimulatory complexes are particularly suitable for serving as adjuvants and / or peptide immunogen stabilizers. The immunostimulatory complexes are in the form of microparticles that can efficiently present the PCSK9 peptide immunogen to cells of the immune system to generate an immune response. The immunostimulatory complexes can be formulated as suspensions for parenteral administration. The immunostimulatory complexes can be formulated in the form of water-in-oil (w / o) emulsions, as suspensions combined with inorganic salts, or as suspensions combined with in situ gelling polymers for efficient delivery of the PCSK9 peptide immunogen construct to cells of the host's immune system after parenteral administration.

[0102] A stabilized immunostimulatory complex can be formed by complexing a PCSK9 peptide immunogen construct with an anionic molecule, an oligonucleotide, a polynucleotide, or a combination thereof via electrostatic binding. The stabilized immunostimulatory complex can be incorporated into a pharmaceutical composition as an immunogen delivery system.

[0103] In certain embodiments, PCSK9 peptide immunogen constructs are designed to contain cationic moieties that are positively charged at a pH in the range of 5.0 to 8.0. The net charge on the cationic moiety of a PCSK9 peptide immunogen construct or a mixture of constructs is calculated by assigning a +1 charge to each lysine (K), arginine (R), or histidine (H), a -1 charge to each aspartic acid (D) or glutamic acid (E), and a 0 charge to other amino acids in the sequence. The charges are summed within the cationic moiety of the PCSK9 peptide immunogen construct and expressed as a net average charge. Suitable peptide immunogens have cationic moieties with an average net positive charge of +1. Preferably, peptide immunogens have a net positive charge in the range of greater than +2. In some embodiments, the cationic moiety of a PCSK9 peptide immunogen construct is a heterologous spacer. In certain embodiments, the cationic moiety of the PCSK9 peptide immunogen construct has a +4 charge when the spacer sequence is (α,ε-N)Lys, (α,ε-N)-Lys-Lys-Lys-Lys (SEQ ID NO: 11), or Lys-Lys-Lys-ε-N-Lys (SEQ ID NO: 12).

[0104] As used herein, the term "anionic molecule" refers to any molecule that is negatively charged at a pH between 5.0 and 8.0. In certain embodiments, the anionic molecule is an oligomer or polymer. The net negative charge on the oligomer or polymer is calculated by assigning a charge of -1 to each phosphodiester or phosphorothioate group in the oligomer. Suitable anionic oligonucleotides are single-stranded DNA molecules containing 8 to 64 nucleotide bases and 1 to 10 repeats of the CpG motif. Preferably, the CpG immunostimulatory single-stranded DNA molecule contains 18 to 48 nucleotide bases and 3 to 8 repeats of the CpG motif.

[0105] More preferably, the anionic oligonucleotide is 5'X1 CGX 2 3', where C and G are unmethylated and X 1 is selected from the group consisting of A (adenine), G (guanine), and T (thymine); X 2 is C (cytosine) or T (thymine). Alternatively, the anionic oligonucleotide may have a 5'(X 3 )2CG(X 4 ) 23′, wherein C and G are not methylated, and X 3 is selected from the group consisting of A, T, or G; X 4 is C or T. In certain embodiments, the CpG oligonucleotide has the sequence: CpG1: 5'TCg TCg TTT TgT CgT TTT gTC gTT TTg TCg TT 3' (fully phosphorothioated) (SEQ ID NO: 108), CpG2: 5' phosphate TCg TCg TTT TgT CgT TTT gTC gTT 3' (fully phosphorothioated) (SEQ ID NO: 109), or CpG3 5'TCg TCg TTT TgT CgT TTT gTC gTT 3' (fully phosphorothioated) (SEQ ID NO: 110).

[0106] The resulting immunostimulating complexes are in the form of particles, typically ranging in size from 1 to 50 microns, and are influenced by many factors, including the relative charge stoichiometry and molecular weight of the interacting species. Particulate immunostimulating complexes have the advantage of providing adjuvant effects and upregulation of specific immune responses in vivo. Furthermore, stabilized immunostimulating complexes are suitable for preparation of pharmaceutical compositions by a variety of processes, including water-in-oil emulsions, mineral salt suspensions, and polymer gels.

[0107] The present disclosure is also directed to pharmaceutical compositions, including formulations, for preventing and / or treating patients with PCSK9-mediated disorders, including elevated serum levels of low-density lipoprotein cholesterol (LDL-C) and CV events. In some embodiments, the pharmaceutical compositions comprise stabilized immunostimulatory complexes (formed via electrostatic association by mixing a peptide composition comprising a mixture of PCSK9 peptide immunogen constructs (e.g., SEQ ID NOS: 65-107) with CpG oligomers), which further enhance the immunogenicity of the PCSK9 peptide immunogen constructs and induce antibodies directed against the PCSK9 / LDL-R receptor-binding region that cross-react with the full-length PCSK9 protein of SEQ ID NO: 1.

[0108] In yet other embodiments, the pharmaceutical composition comprises a mixture of PCSK9 peptide immunogen constructs (e.g., any combination of SEQ ID NOs: 65-107) in the form of a stabilized immunostimulatory complex with a CpG oligomer (optionally mixed with an inorganic salt (including ALHYDROGEL or ADJUPHOS) as an adjuvant with a high safety factor) to form a suspension formulation for administration to a host.

[0109] antibody The present disclosure also provides antibodies induced by the PCSK9 peptide immunogen constructs.

[0110] The present disclosure provides a PCSK9 peptide immunogen construct and formulations thereof that are cost-effective to produce, optimally designed, capable of inducing high-titer antibodies that target the catalytic domain of the PCSK9 molecule (e.g., SEQ ID NOS: 2-9), more specifically the PCSK9 and LDL-R receptor binding region (e.g., SEQ ID NOS: 2-6 and 8-9), capable of breaking immune tolerance to the self-protein PCSK9, and resulting in a high responder rate in immunized hosts. The antibodies generated by the PCSK9 peptide immunogen construct have high affinity for the PCSK9 / LDL-R receptor binding region.

[0111] In some embodiments, PCSK9 peptide immunogen constructs for inducing antibodies comprise a hybrid of a PCSK9 peptide targeting the catalytic domain of PCSK9, including the LDL-R receptor binding region (e.g., SEQ ID NOS: 2-9), and a heterologous Th epitope (SEQ ID NOS: 13-64) derived from a pathogenic protein (such as the measles virus fusion (MVF) protein and others), optionally linked via a spacer. The B cell epitope and Th epitope peptide of the PCSK9 peptide immunogen construct work together to stimulate the generation of highly specific antibodies that cross-react with the catalytic domain (SEQ ID NOS: 111) of the full-length PCSK9 protein (SEQ ID NOS: 1).

[0112] Traditional methods for enhancing the immunogenicity of peptides, such as chemical coupling to carrier proteins (e.g., keyhole limpet hemocyanin (KLH) or other carrier proteins such as diphtheria toxoid (DT) and tetanus toxoid (TT) proteins), typically result in the generation of large amounts of antibodies directed against the carrier protein. Thus, a major drawback of such peptide-carrier protein compositions is that most (>90%) of the antibodies generated by the immunogen are non-functional antibodies directed against the carrier protein (KLH, DT, or TT), which can lead to epitopic suppression.

[0113] Unlike traditional methods for enhancing peptide immunogenicity, antibodies generated by the PCSK9 peptide immunogen constructs of the present disclosure (e.g., SEQ ID NOS: 65-107) bind highly specifically to the catalytic domain of the PCSK9 B cell epitope peptide (SEQ ID NOS: 2-9), with few, if any, antibodies directed against heterologous Th epitopes (e.g., SEQ ID NOS: 13-64) or optional heterologous spacers.

[0114] Based on their unique characteristics and properties, the antibodies of the present disclosure induced by the PCSK9 peptide immunogen constructs have the potential to provide a prophylactic immunotherapeutic approach to prevent and / or treat PCSK9-mediated disorders in subjects, including elevated serum levels of low-density lipoprotein cholesterol (LDL-C) and CV events.

[0115] method The present disclosure is also directed to methods of preparing and using the PCSK9 peptide immunogenic constructs, compositions, and pharmaceutical compositions.

[0116] a. Methods for Producing PCSK9 Peptide Immunogen Constructs The PCSK9 peptide immunogen constructs of the present disclosure can be prepared by chemical synthesis methods familiar to those skilled in the art (see, for example, Fields, GB, et al., 1992). PCSK9 peptide immunogen constructs can be synthesized using automated Merrifield solid-phase synthesis techniques, in which side-chain protected amino acids are used to protect the α-NH2 with either t-Boc or F-moc chemistry, and this technique is performed, for example, on an Applied Biosystems Peptide Synthesizer, Model 430A or Model 431. Preparation of PCSK9 peptide immunogen constructs containing combinatorial library peptides of Th epitopes can be achieved by using a mixture of alternative amino acids for coupling at a given variable position.

[0117] After the desired PCSK9 peptide immunogen construct is fully assembled, the peptide can be cleaved from the resin and the functional groups on the amino acid side chains can be deprotected by treating the resin according to standard procedures. The free peptide can be purified by HPLC and biochemically characterized. This characterization can be performed, for example, by amino acid analysis or sequencing. Methods for purifying and characterizing peptides are well known to those skilled in the art.

[0118] The quality of the peptides produced by this chemical process can be controlled and defined, and as a result, reproducibility of the PCSK9 peptide immunogen construct, immunogenicity, and yield can be ensured. A detailed description of the production of PCSK9 peptide immunogen constructs via solid-phase peptide synthesis is provided in Example 1.

[0119] The range of structural variability that allows for retention of the desired immunological activity has been shown to be much more flexible than the range of structural variability that allows for retention of specific drug activity with small molecule drugs, or the range of structural variability that allows for retention of the desired activity and undesired toxicity seen with large molecules that occur with biologically derived drugs.

[0120] Thus, peptide analogs, whether intentionally designed or inevitably resulting from errors in the synthetic process as a mixture of deleted sequence by-products with similar chromatographic and immunological properties to the intended peptide, are often as effective as purified preparations of the desired peptide. Designed analogs and unintentional analog mixtures are effective so long as discriminatory QC procedures are developed to monitor both the manufacturing and product evaluation processes to ensure the reproducibility and potency of final products using such peptides.

[0121] PCSK9 peptide immunogenic constructs can also be prepared using recombinant DNA technology, including nucleic acid molecules, vectors, and / or host cells. Thus, nucleic acid molecules encoding PCSK9 peptide immunogenic constructs and nucleic acid molecules encoding immunologically functional analogs of peptide immunogenic constructs are also encompassed by the present disclosure as part of the present invention. Similarly, vectors (including expression vectors) containing nucleic acid molecules and host cells containing such vectors are also encompassed by the present disclosure as part of the present invention.

[0122] Various exemplary embodiments also encompass methods for producing PCSK9 peptide immunogen constructs and their immunologically functional analogs. For example, the method may include incubating host cells containing expression vectors comprising nucleic acid molecules encoding the PCSK9 peptide immunogen constructs and / or their immunologically functional analogs under conditions for expression of the peptides and / or analogs. Longer synthetic peptide immunogens can be synthesized using well-known recombinant DNA techniques. Such techniques, along with detailed protocols, are provided in well-known standard manuals. To construct a gene encoding a peptide of the invention, a nucleic acid sequence encoding the amino acid sequence is obtained by reverse-translating the amino acid sequence, and such nucleic acid sequence preferably uses optimal codons for the organism in which the gene will be expressed. A synthetic gene is then typically prepared by synthesizing oligonucleotides encoding the peptide and any necessary control elements. The synthetic gene is inserted into an appropriate cloning vector and transfected into a host cell. The peptide is then expressed under appropriate conditions for the selected expression system and host. The peptide is purified and characterized by standard methods.

[0123] b. Methods for producing immunostimulating complexes Various exemplary embodiments also encompass methods for producing immunostimulatory complexes (ISCs) containing PCSK9 peptide immunogen constructs and CpG oligodeoxynucleotide (ODN) molecules. Stabilized immunostimulatory complexes (ISCs) are obtained from the cationic portion of the PCSK9 peptide immunogen construct and polyanionic CpG ODN molecules. This self-assembly system is facilitated by electrostatic charge neutralization. The degree of association is determined by the stoichiometry of the molar charge ratio of the cationic portion of the PCSK9 peptide immunogen construct to the anionic oligomer. The non-covalent electrostatic association of the PCSK9 peptide immunogen construct with the CpG ODN is a completely reproducible process. The peptide / CpG ODN immunostimulatory complex assembly promotes presentation to "professional" antigen-presenting cells (APCs) of the immune system, thereby further enhancing its own immunogenicity. Such complexes are easily characterized for quality control during manufacturing. The peptide / CpG ISCs are well tolerated in vivo. This novel microparticle system containing CpG ODN and PCSK9 peptide immunogen constructs was designed to take advantage of the general B cell mitogenicity associated with the use of CpG ODN, and also to promote a balanced Th-1 / Th-2 type response.

[0124] The CpG ODN in the pharmaceutical composition of the present disclosure is 100% bound to immunogens in a process mediated by electrostatic neutralization of opposite charges, resulting in the formation of micron-sized particles.This microparticle form can significantly reduce the CpG dose compared to the conventional use of CpG adjuvants, reducing the possibility of harmful innate immune responses, and promotes alternative immunogen processing pathways involving antigen-presenting cells (APCs).Therefore, such formulations are conceptually novel and are expected to bring benefits by promoting the stimulation of immune responses through alternative mechanisms.

[0125] c. Methods for producing pharmaceutical compositions Various exemplary embodiments also encompass pharmaceutical compositions comprising the PCSK9 peptide immunogenic constructs. In certain embodiments, the pharmaceutical compositions may be water-in-oil emulsions or suspensions containing inorganic salts.

[0126] For pharmaceutical compositions to be used by large populations, safety is another important consideration. Although water-in-oil emulsions have been used in many clinical trials, alum remains the primary adjuvant used in formulations due to its safety. Therefore, alum or its inorganic salt (aluminum phosphate (ADJUPHOS)) is often used as an adjuvant in preparations for clinical application.

[0127] Other adjuvants and immunostimulants include 3-O-deacylated monophosphoryl lipid A (MPL) or 3-DMP, polymeric or monomeric amino acids (such as polyglutamic acid or polylysine), etc. Such adjuvants may be used with or without other specific immunostimulants. Such other specific immunostimulants include muramyl peptides (e.g., N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), N-acetylglucosaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) Theramide™), or other bacterial cell wall components. Oil-in-water emulsions include MF59 (see WO 90 / 14837 to Van Nest et al., which is incorporated herein by reference in its entirety) (5% squalene, 0.5% TWEEN 80, and 0.5% Span 85 (optionally with various amounts of MTP-PE) formulated into submicron particles using a microfluidizer), SAF (10% squalene, 0.4% TWEEN 80, 5% pluronic block polymer L121, and thr-MDP, which are microfluidized into submicron emulsions or vortexed to produce larger particle size emulsions), and Ribi™ Adjuvant System (RAS) (Ribi ImmunoChem, Hamilton, Mont.) (2% squalene, 0.2% TWEEN 80, and one or more bacterial cell wall components selected from the group consisting of monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS) (preferably MPL+CWS (Detox™)).Other adjuvants include complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA), and cytokines such as interleukins (IL-1, IL-2, and IL-12), macrophage colony-stimulating factor (M-CSF), and tumor necrosis factor (TNF-α).

[0128] The choice of adjuvant depends on the stability of the immunogenic formulation containing the adjuvant, the route of administration, the dosing schedule, and the efficacy of the adjuvant for the species being immunized; in humans, a pharmaceutically acceptable adjuvant is one that has been or can be approved for administration to humans by the relevant regulatory agency. For example, alum, MPL, or incomplete Freund's adjuvant (Chang, JCC, et al., 1998) (which is incorporated herein by reference in its entirety) are all suitable for administration to humans, either alone or in optional combination.

[0129] The composition may contain a pharmaceutically acceptable and nontoxic carrier or diluent, which is defined as a medium commonly used in formulating pharmaceutical compositions for administration to animals or humans. The diluent is selected so as not to affect the biological activity of the compound to which it is combined. Examples of such diluents include distilled water, phosphate-buffered saline, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also contain other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers, and the like.

[0130] Pharmaceutical compositions may also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acid, polyglycolic acid, and copolymers (e.g., latex-functionalized sepharose, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (e.g., oil droplets or liposomes). Additionally, such carriers may function as immunostimulating agents (i.e., adjuvants).

[0131] The pharmaceutical compositions of the present invention may further comprise a suitable delivery vehicle, including, but not limited to, viruses, bacteria, biodegradable microspheres, microparticles, nanoparticles, liposomes, collagen minipellets, and cochleate vesicles.

[0132] d. Methods of using pharmaceutical compositions The present disclosure also includes methods of using pharmaceutical compositions comprising PCSK9 peptide immunogenic constructs.

[0133] In certain embodiments, pharmaceutical compositions comprising PCSK9 peptide immunogenic constructs may be used to treat patients with PCSK9-mediated disorders, including elevated serum levels of low-density lipoprotein cholesterol (LDL-C) and CV events.

[0134] In some embodiments, the method comprises administering to a host in need thereof a pharmaceutical composition comprising a pharmacologically effective amount of a PCSK9 peptide immunogenic construct. In certain embodiments, the method comprises administering to a warm-blooded animal (e.g., a human, a cynomolgus monkey, a mouse) a pharmaceutical composition comprising a pharmacologically effective amount of a PCSK9 peptide immunogenic construct, thereby inducing highly specific antibodies that cross-react with the full-length human PCSK9 protein (SEQ ID NO: 1).

[0135] In certain embodiments, pharmaceutical compositions comprising PCSK9 peptide immunogenic constructs may be used to treat PCSK9-mediated disorders, including elevated serum levels of low-density lipoprotein cholesterol (LDL-C) and CV events in a subject.

[0136] e. In vitro functional assays and in vivo proof-of-concept studies Antibodies induced in hosts immunized with PCSK9 peptide immunogen constructs can be used in in vitro functional assays and in in vivo efficacy tests. Such functional assays or tests include, but are not limited to, the following: a. generating highly specific antibodies that can inhibit PCSK9 and LDL-R receptor binding along with the associated downstream cellular events that lead to LDL-R degradation, thereby causing increased LDL-C uptake by LDL-R-expressing cells; and b. Reducing the levels of LDL-C and T-CHO in the plasma of immunized hosts.

[0137] The PCSK9 peptide immunogen constructs and formulations thereof of the present disclosure can effectively function as pharmaceutical compositions to prevent and / or treat subjects susceptible to or suffering from PCSK9-mediated disorders, including elevated serum levels of low-density lipoprotein cholesterol (LDL-C) and CV events in the subject.

[0138] Specific Embodiments (1) A PCSK9 peptide immunogenic construct having about 20 or more amino acids, wherein the PCSK9 peptide immunogenic construct has the following formula: (Th) m -(A) n -(PCSK9 functional B cell epitope peptide)-X or (PCSK9 functional B cell epitope peptide)-(A) n -(Th) m -X or (Th) m -(A) n -(PCSK9 functional B cell epitope peptide)-(A) n -(Th) m -X is expressed as During the ceremony, Th is a heterologous helper T cell epitope; A is a heterologous spacer, (PCSK9 functional B cell epitope peptide) is a B cell epitope peptide having 7 to about 30 amino acid residues derived from the catalytic domain of PCSK9 protein (SEQ ID NO: 111), X is the α-COOH or α-CONH2 of an amino acid, m is from 1 to about 4; The PCSK9 peptide immunogen construct, wherein n is 0 to about 10.

[0139] (2) The PCSK9 peptide immunogen construct according to (1), wherein the PCSK9 functional B-cell epitope peptide is selected from the group consisting of SEQ ID NOs: 2 to 9.

[0140] (3) The PCSK9 peptide immunogen construct according to (1), wherein the Th epitope is selected from the group consisting of SEQ ID NOs: 13 to 64.

[0141] (4) The PCSK9 peptide immunogen construct according to (1), wherein the PCSK9 functional B cell epitope peptide is selected from the group consisting of SEQ ID NOs: 2 to 9, and the Th epitope is selected from the group consisting of SEQ ID NOs: 13 to 64.

[0142] (5) The PCSK9 peptide immunogen construct according to (1), wherein the peptide immunogen construct is selected from the group consisting of SEQ ID NOs: 65 to 107.

[0143] (6) a. a B cell epitope comprising about 7 to about 30 amino acid residues derived from the catalytic domain of the PCSK9 sequence of SEQ ID NO: 111; b. A helper T cell epitope comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 64, and any combination thereof; c. an optional heterologous spacer selected from the group consisting of the amino acids Lys-, Gly-, Lys-Lys-Lys-, (α,ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 11), Lys-Lys-Lys-ε-N-Lys (SEQ ID NO: 12), and Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO: 10), and any combination thereof; A PCSK9 peptide immunogen construct comprising: The PCSK9 peptide immunogen construct, wherein the B cell epitope is covalently linked to the helper T cell epitope either directly or via the optional heterologous spacer.

[0144] (7) The PCSK9 peptide immunogen construct according to (6), wherein the B cell epitope is selected from the group consisting of SEQ ID NOs: 2 to 9.

[0145] (8) The PCSK9 peptide immunogen construct of (6), wherein the optional heterologous spacer is (α,ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 11), Lys-Lys-Lys-ε-N-Lys (SEQ ID NO: 12), or Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO: 10), wherein Xaa is any amino acid.

[0146] (9) The PCSK9 peptide immunogen construct according to (6), wherein the helper T cell epitope is covalently linked to the amino or carboxyl terminus of the B cell epitope.

[0147] (10) The PCSK9 peptide immunogen construct of (6), wherein the helper T cell epitope is covalently linked to the amino or carboxyl terminus of the B cell epitope via the optional heterologous spacer.

[0148] (11) A composition comprising the PCSK9 peptide immunogen construct described in (1).

[0149] (12) a. The PCSK9 peptide immunogen construct described in (1); b. a pharmaceutically acceptable delivery vehicle and / or adjuvant; A pharmaceutical composition comprising:

[0150] (13) a. The PCSK9 functional B-cell epitope peptide is selected from the group consisting of SEQ ID NOs: 2 to 9; b. the Th epitope is selected from the group consisting of SEQ ID NOs: 13-64; c. the heterologous spacer is selected from the group consisting of the amino acids Lys-, Gly-, Lys-Lys-Lys-, (α,ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 11), Lys-Lys-Lys-ε-N-Lys (SEQ ID NO: 12), and Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO: 10), and any combination thereof; The pharmaceutical composition according to (12), wherein the PCSK9 peptide immunogen construct is mixed with a CpG oligodeoxynucleotide (ODN) to form a stabilized immunostimulatory complex.

[0151] (14) a. The PCSK9 peptide immunogen construct is selected from the group consisting of SEQ ID NOs: 65 to 107; The pharmaceutical composition according to (12), wherein the PCSK9 peptide immunogen construct is mixed with a CpG oligodeoxynucleotide (ODN) to form a stabilized immunostimulatory complex.

[0152] (15) A method for producing an antibody against PCSK9 in an animal, the method comprising administering to the animal the pharmaceutical composition according to (12).

[0153] (16) An isolated antibody or epitope-binding fragment thereof that specifically binds to the PCSK9 and LDL-R receptor binding domains of SEQ ID NOs: 2 to 9.

[0154] (17) The isolated antibody or epitope-binding fragment thereof according to (16), bound to the PCSK9 peptide immunogen construct.

[0155] (18) A composition comprising the isolated antibody or epitope-binding fragment thereof according to (16).

[0156] (19) A method for preventing and / or treating a patient having a PCSK9-mediated disorder, including increased serum levels of low-density lipoprotein cholesterol (LDL-C) and CV events, in an animal, the method comprising administering to the animal the pharmaceutical composition according to (12). [Example]

[0157] Example 1 Synthesis of PCSK9-related peptides and preparation of their formulations a. Synthesis of PCSK9-related peptides We describe the synthesis of PCSK9-related peptides that were part of the development effort for PCSK9 peptide immunogen constructs. These peptides were synthesized in small-scale quantities useful for serological assays, laboratory pilot studies, and field trials, as well as in large-scale (kilogram) quantities useful for industrial / commercial production of pharmaceutical compositions. A large repertoire of PCSK9-related antigenic peptides with sequences ranging in length from approximately 10 to 70 amino acids was designed for the purposes of epitope mapping and screening and selection of peptide immunogen constructs most suitable for use in an effective PCSK9-targeted therapeutic vaccine.

[0158] Representative full-length human PCSK9 (SEQ ID NO: 1) and PCSK9 peptide fragments are listed in Table 1 (SEQ ID NOs: 2-9).

[0159] PCSK9 peptide immunogen constructs were prepared by synthetically linking selected PCSK9 B cell epitope peptides to carefully designed helper T cell (Th) epitope peptides (shown in Table 2 (SEQ ID NOS: 13-64)) derived from pathogen proteins. Pathogen proteins from which these Th epitope peptides were derived include measles virus fusion protein (MVF), hepatitis B surface antigen protein (HBsAg), influenza, and Clostridium tetani. These Th epitope peptides include those from Epstein-Barr virus (EBV), Streptococcus aureus (STI), Mycobacterium tuberculosis (MT ...

[0160] Table 3 shows representative PCSK9 peptide immunogen constructs (SEQ ID NOS: 65-107) selected from hundreds of peptide constructs. All peptides used in immunogenicity studies or related serological studies to detect and / or measure anti-PCSK9 antibodies were synthesized on small scales using F-moc chemistry on Applied BioSystems Model 430A, Model 431, and / or Model 433 peptide synthesizers. Each peptide was generated by independent synthesis on a solid support, with the N-terminus protected by F-moc and side-chain protecting groups for trifunctional amino acids. Completed peptides were cleaved from the solid support, and the side-chain protecting groups were removed using 90% trifluoroacetic acid (TFA). Synthetic peptide preparations were evaluated by matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry to confirm the correct amino acid content. Each synthetic peptide was also evaluated by reverse-phase HPLC (RP-HPLC) to confirm the synthesis profile and concentration of the preparations. Despite strict control of the synthetic process (including stepwise monitoring of coupling efficiency), peptide analogs were also generated due to unintended events during the elongation cycle, including amino acid insertions, deletions, substitutions, and premature terminations. Thus, synthesized preparations typically contained multiple peptide analogs in addition to the targeted peptide.

[0161] Despite the presence of such unintended peptide analogs, the resulting synthetic peptide preparations were nevertheless suitable for use in immunological applications, including immunodiagnostics (as antibody capture antigens) and pharmaceutical compositions (as peptide immunogens). Typically, such peptide analogs, whether intentionally designed or arising through the synthetic process as a mixture of by-products, are often as effective as purified preparations of the desired peptide, so long as distinguishing QC procedures are developed to monitor both the manufacturing and product evaluation processes to ensure the reproducibility and potency of final products using such peptides. Large-scale peptide synthesis in hundreds of grams to kilogram quantities can be performed on a customized automated peptide synthesizer, UBI2003 or similar, at the 15 mmol to 150 mmol scale.

[0162] For the active ingredient to be used in the final pharmaceutical composition for clinical trials, the PCSK9 peptide immunogen construct was purified by preparative RP-HPLC with shallow gradient elution and characterized for purity and identity by MALDI-TOF mass spectrometry, amino acid analysis, and RP-HPLC.

[0163] b. Preparation of Compositions Comprising PCSK9 Peptide Immunogen Constructs Formulations using water-in-oil emulsions and formulations in suspensions containing inorganic salts have been prepared. When designing pharmaceutical compositions for use by large populations, safety is another important consideration. Despite the fact that water-in-oil emulsions have been used in humans as pharmaceutical compositions in many clinical trials, alum remains the primary adjuvant for use in pharmaceutical compositions due to its safety. Therefore, alum or its inorganic salt (ADJUPHOS (aluminum phosphate)) is often used as an adjuvant in preparations for clinical application.

[0164] Briefly, the formulations specified in each of the study groups described below generally included all types of designer PCSK9 peptide immunogen constructs. Over 40 peptide immunogen constructs were carefully evaluated in guinea pigs for their relative immunogenicity to the corresponding PCSK9 peptide used as the B-cell epitope within the construct.

[0165] Varying amounts of the PCSK9 peptide immunogen construct were prepared as a water-in-oil emulsion using SEPPIC MONTANIDE™ ISA51, an oil approved for human use, or mixed with the inorganic salts ADJUPHOS (aluminum phosphate) or ALHYDROGEL (alum) (as specified). Typically, compositions were prepared by dissolving the PCSK9 peptide immunogen construct in water at approximately 20-2,000 μg / mL, and then formulated as a water-in-oil emulsion using MONTANIDE™ ISA51 (1:1 by volume) or with the inorganic salts ADJUPHOS or ALHYDROGEL (alum) (1:1 by volume). The compositions were kept at room temperature for approximately 30 minutes and mixed by vortexing for approximately 10-15 seconds before use in immunization. Animals were immunized with two to three doses of a particular composition, administered intramuscularly at weeks post-immunization (wpi) 0 (prime) and 3 (boost), with an optional second boost at 5 or 6 wpi. Sera from the immunized animals were then tested with selected B-cell epitope peptide(s) to assess the immunogenicity of the various PCSK9 peptide immunogen constructs present in the formulations and the cross-reactivity of the corresponding sera with the PCSK9 protein. For PCSK9 peptide immunogen constructs that were found to have potent immunogenicity in initial screening in guinea pigs, the functional properties of the corresponding sera were further tested in in vitro assays. Selected candidate PCSK9 peptide immunogen constructs were then formulated as water-in-oil emulsion-based, inorganic salt-based, and alum-based formulations for the specific dosing regimens indicated by the immunization protocol.

[0166] Only the most promising PCSK9 peptide immunogen constructs were extensively further evaluated before being incorporated into a final formulation for immunogenicity, duration, toxicity, and efficacy studies in GLP-guided preclinical studies in preparation for Investigational New Drug (IND) submission, and subsequent clinical trials in patients suffering from PCSK9-mediated disorders, including elevated serum levels of low-density lipoprotein cholesterol (LDL-C) and CV events.

[0167] The following examples are intended to illustrate the present invention and should not be used to limit the scope of the invention.

[0168] Example 2 Serological Assays and Reagents Detailed below are serological assays and reagents for assessing the functional immunogenicity of PCSK9 peptide immunogen constructs and formulations thereof.

[0169] a. PCSK9-based or PCSK9 B-cell epitope peptide-based ELISA assays to analyze immunogenicity and antibody specificity An ELISA assay was developed to evaluate the immune serum samples described in the Examples below, and is described below: Wells of a 96-well plate were individually coated with 100 μL of 2 μg / mL (unless otherwise specified) PCSK9 or a PCSK9 B-cell epitope peptide (e.g., SEQ ID NOS: 2-9) in 10 mM NaHCO3 buffer (pH 9.5) (unless otherwise specified) for 1 hour at 37°C.

[0170] The wells coated with PCSK9 or PCSK9 B cell epitope peptide were incubated with 250 μL of PBS containing 3% gelatin by weight at 37° C. for 1 hour to block nonspecific protein binding sites, and then washed three times with PBS containing 0.05% TWEEN® 20 by volume and dried. The serum to be analyzed was diluted 1:20 (unless otherwise specified) with PBS containing 20% ​​normal goat serum by volume, 1% gelatin by weight, and 0.05% TWEEN® 20 by volume. 100 μL of the diluted sample (e.g., serum, plasma) was added to each well and incubated at 37° C. for 60 minutes. Next, the wells were washed six times with PBS containing 0.05% TWEEN® 20 by volume to remove unbound antibodies. Horseradish peroxidase (HRP)-conjugated species-specific (e.g., guinea pig or rat) goat polyclonal anti-IgG antibody or Protein A / G was used as a labeled tracer to bind to the antibody / peptide antigen complex formed in positive wells. 100 μL of HRP-labeled detection reagent, optimally diluted and previously titrated in PBS containing 1% (v / v) normal goat serum and 0.05% (v / v) TWEEN® 20, was added to each well and incubated for an additional 30 minutes at 37°C. Unbound antibody was removed by washing the wells six times with PBS containing 0.05% (v / v) TWEEN® 20, and the wells were incubated for an additional 15 minutes with 100 μL of a substrate mixture containing 0.04% (v / v) 3',3',5',5'-tetramethylbenzidine (TMB) and 0.12% (v / v) hydrogen peroxide in sodium citrate buffer. This substrate mixture was used to detect the peroxidase label by forming a colored product. The reaction was stopped by adding 100 μL of 1.0 M H2SO4 and measuring the absorbance (A) at 450 nm. 450 For antibody titers of vaccinated animals administered various peptide vaccine formulations, serum was tested in 10-fold serial dilutions ranging from 1:100 to 1:10,000 dilutions or in 4-fold serial dilutions ranging from 1:100 to 1:4.19 × 10 8 The serum was tested in dilutions. The titer (Log 10) is expressed as A 450 A cutoff value is set to 0.5. 450 was calculated by linear regression analysis.

[0171] b. Evaluation of antibody reactivity to Th peptides by Th peptide-based ELISA test Wells of a 96-well plate were individually coated with 100 μL of 2 μg / mL (unless otherwise specified) Th peptide in 10 mM NaHCO3 buffer (pH 9.5) (unless otherwise specified) for 1 hour at 37°C, and subjected to the same ELISA method as above. ELISA was performed as described above. To determine antibody titers from vaccinated animals administered various PCSK9 peptide vaccine formulations, sera were tested in 10-fold serial dilutions ranging from 1:100 to 1:10,000 dilutions. The titers (Log ) of the tested sera were calculated. 10 ) is expressed as A 450 A cutoff value is set to 0.5. 450 was calculated by linear regression analysis.

[0172] Detailed specificity analysis of target PCSK9 B cell epitope peptides determined by epitope mapping with a cB cell epitope cluster 10-mer peptide-based ELISA assay Detailed specificity analysis of anti-PCSK9 antibodies obtained from hosts immunized with PCSK9 peptide immunogen constructs can be performed by epitope mapping using B cell epitope cluster 10-mer peptide-based ELISA tests. Briefly, individual PCSK9 10-mer peptides can be used to coat the wells of a 96-well plate at 0.5 μg / 0.1 mL per well. Then, 100 μL of serum sample (1:100 dilution in PBS) can be incubated in duplicate in the 10-mer plate wells according to the steps of the antibody ELISA method described above. Target B cell epitope specificity analysis of anti-PCSK9 antibodies obtained from immunized hosts can be performed using the corresponding PCSK9 peptide or an unrelated control peptide to confirm specificity.

[0173] d. Immunogenicity evaluation Pre-immune and immune serum samples were collected from animal or human subjects according to the experimental vaccination protocol and heated at 56°C for 30 minutes to inactivate serum complement factors. After administration of the vaccine formulation, blood samples were collected according to the protocol, and their immunogenicity against the specific target site(s) was assessed by a corresponding PCSK9 B-cell epitope peptide-based ELISA test. Serially diluted sera were tested, and the Log of the reciprocal dilution factor was used. 10 Positive titers were expressed as a positive titer. The immunogenicity of a particular vaccine formulation is assessed for its ability to elicit a high-titer antibody response directed to a desired epitope within the target antigen with specificity and high cross-reactivity with the PCSK9 protein, while maintaining low to negligible antibody reactivity to the helper T cell epitopes used to boost the desired B cell response.

[0174] Example 3 Methods for functional characterization of antibodies in in vitro assays for LDL-C uptake and measurement of in vivo serum / plasma levels of LDL-C and T-CHO Purified anti-PCSK9 antibodies in immune sera or immunization vaccines were further tested for their ability to inhibit PCSK9 binding to the LDL-R receptor as reflected by LDL-C uptake by LDL-R-expressing cell lines.

[0175] a. Antibody purification All antibody purification procedures were performed according to the manual of the antibody purification kit (Thermo Fisher, Cat. No. 89953). The concentration of each purified IgG was carefully calibrated for each group for use in in vitro assays.

[0176] b. Cell Preparation and Maintenance The HepG2 cell line was purchased from the American Type Culture Collection (Manassas, VA) and maintained in DMEM medium supplemented with 10% fetal bovine serum (FBS), 4.5 g / L L-glutamine, sodium pyruvate, and 1% penicillin / streptomycin in a humidified incubator at 37°C with 5% CO2.

[0177] c. Cell-based LDL-C uptake assay Human HepG2 cells were cultured at a density of 50,000 cells / well in DMEM medium supplemented with 10% FBS in black, clear-bottom 96-well microplates. Cells were incubated at 37°C for 48 hours. Cells were then starved overnight with 0.3% BSA DMEM. To form PCSK9 and antibody-PCSK9 immune complexes with purified antibodies from guinea pig pre-immune or immune serum, 5 μg / mL human PCSK9 (Biolegend, #592506) was incubated with various concentrations of purified guinea pig polyclonal antibodies serially diluted in either uptake buffer (DMEM containing 0.3% FBS) or uptake buffer alone (control) for 1 hour at room temperature. After washing the cells with PBS, the PCSK9 / antibody mixture was transferred to the cells in the 96-well plate, followed by the transfer of LDL-BODIPY (Invitrogen) diluted in uptake buffer to a final concentration of 5 μg / mL. After incubation at 37°C for 2 hours, the cells were thoroughly washed with PBS, and the fluorescent signals of the cells were detected at 480–520 nm (excitation) and 520–600 nm (emission) using a SpectraMax i3x reader (Molecular Devices) as the measurement parameters for LDL-C uptake by human HepG2 cells.

[0178] d. Measurement of serum / plasma LDL-C and T-CHO levels in guinea pigs Plasma LDL-cholesterol (LDL-C) and total cholesterol (T-CHO) levels were measured for each blood sample from each animal using a Hitachi 7080 analyzer with the Wako L-type CHO M kit (catalog no. 462-12491) and Roche L-type LDL-C kit (catalog no. 137520), respectively, according to the manufacturer's instructions. Dilutions of cholesterol / LDL standards or test samples (70 μL per sample volume) were added to each well of a 96-well plate. One hundred forty microliters (140 μL) of prepared LDL-C reagent was added as a calibrator. The plate was incubated at 37°C for 5 minutes, and the absorbance of the developed color was read at 600 nm within 30 minutes.

[0179] Example 4 Animals used in safety, immunogenicity, toxicity, and efficacy studies A guinea pig Immunogenicity studies were conducted in adult, naive, and adult male and female Duncan-Hartley guinea pigs (300-350 g / BW). Experiments utilized at least three guinea pigs per group. Duncan-Hartley guinea pigs (8-12 weeks old, Covance Research Laboratories, Denver, PA, USA) and the associated protocols were performed in a UBI-sponsored contract animal facility under approved IACUC procedures.

[0180] b. Immunization of guinea pigs with placebo and formulations containing PCSK9 peptide immunogen constructs Guinea pigs were used for immunization, with three animals per group. Experimental groups were immunized intramuscularly with a PSCK9 peptide immunogen construct formulated with ISA51 and CpG at a dose of 400 μg / 1.0 mL for the prime and boost immunizations, respectively. Administration generally occurred at 0, 3, 6, 9, 12, and 15 WPI, for a total of five doses. All animals had free access to mouse chow and water. Animals were bled generally at 0, 3, 6, 9, 12, and 15 WPI. All animals were fasted for 12 hours before blood collection. Blood samples were collected for titration against PCSK9 B-cell epitope peptides or full-length recombinant PCSK9 protein. Plasma LDL-C and T-CHO levels in each blood sample were also measured by standard blood testing procedures for component analysis.

[0181] Example 5 Vaccine formulation for immunogenicity evaluation of PCSK9 peptide immunogen constructs in guinea pigs Below is a more detailed description of the pharmaceutical compositions and vaccine formulations used in each experiment.

[0182] Briefly, the formulations specified in each test group generally included all types of designer PCSK9 peptide immunogen constructs containing segments of PCSK9 B-cell epitope peptides linked via different types of spacers (e.g., εLys (εK) or lysine-lysine-lysine (KKK)) to enhance the solubility of the peptide constructs, and non-selective helper T-cell epitopes (including two sets of artificial helper T-cell epitopes derived from measles virus fusion protein and hepatitis B surface antigen). The PCSK9 B-cell epitope peptides were linked to the N- or C-terminus of the designer peptide constructs. Several designer PCSK9 peptide immunogen constructs were initially evaluated for their relative immunogenicity in guinea pigs using the corresponding PCSK9 B-cell epitope peptides. Various amounts of PCSK9 peptide immunogen constructs were prepared as water-in-oil emulsions using Seppic MONTANIDE ISA51, an oil approved for human vaccine use, or as suspensions using inorganic salts (ADJUPHOS) or ALHYDROGEL (Alum) (as specified). Formulations were typically prepared by dissolving the PCSK9 peptide construct in water at approximately 20–800 μg / mL and then formulated as water-in-oil emulsions using MONTANIDE ISA51 (1:1 by volume) or inorganic salts (ADJUPHOS) or ALHYDROGEL (Alum) (1:1 by volume). The formulations were held at room temperature for approximately 30 minutes and mixed by vortexing for approximately 10–15 seconds before use in immunization.

[0183] Some animals were immunized with 2 to 5 doses of a particular vaccine formulation, administered at 0 (prime) and 3 (boost) weeks post-immunization (wpi), with an optional second boost administered at 5 or 6 wpi, via the intramuscular route. These immunized animals were then evaluated for the immunogenicity of the corresponding PCSK9 peptide immunogen construct used in each formulation, including its cross-reactivity with the corresponding PCSK9 B-cell epitope peptide or full-length PCSK9. PCSK9 peptide immunogen constructs that were found to be potently immunogenic in initial screening in guinea pigs can be further tested in other organisms as water-in-oil emulsion-based, inorganic salt-based, and alum-based formulations for the specific dosing regimen indicated by the immunization protocol.

[0184] Example 6 Design rationale, screening, identification, functional characterization, and optimization of a multicomponent vaccine formulation incorporating a PCSK9 peptide immunogen construct to treat patients with PCSK9-mediated disorders, including elevated serum levels of low-density lipoprotein cholesterol (LDL-C) and CV events Based on the scientific information provided in Figures 1-4, PCSK9 was selected as the target molecule for designing the peptide immunogen constructs of the present disclosure. Figure 1 illustrates a general overview of the steps using a flow chart that illustrates the development process from discovery to commercialization (industrialization) of a PCSK9 vaccine formulation. Detailed evaluation and analysis of each of these steps has led to the numerous experiments that have thus far resulted in the commercialization of safe and effective PCSK9 peptide immunogen construct-containing pharmaceutical formulations.

[0185] The mechanism and role of PCSK9 in LDL-C metabolism are described in Chaudhary, R., et al., 2017. Figure 2 illustrates the full-length sequence of human PCSK9 (SEQ ID NO: 1), consisting of 692 amino acid residues. The full-length PCSK9 protein consists of a signal peptide (residues 1-30), a prodomain (residues 31-152), a catalytic domain (residues 153-454), and a C-terminal (CT) domain (residues 455-692). Binding of PCSK9 to the EGF-A repeats of the LDL-R is mediated by a patch of residues within the catalytic domain of PCSK9 (SEQ ID NO: 111). The catalytic domain of PCSK9 is involved in both autocatalytic cleavage and binding of PCSK9 to the LDL-R. Figure 3 identifies the amino acid residues on PCSK9 and the LDL-R that bind to each other. These regions within the PCSK9 catalytic domain are around which the disclosed PCSK9 peptide immunogen constructs of the present invention are designed. Figure 4 illustrates sequence alignments of the catalytic domains of PCSK9 from human, monkey, mouse, rat, and guinea pig. These alignments facilitate construct design and allow the selection of analog immunogen constructs to test in target species to demonstrate the ability of designer PCSK9 peptide immunogen constructs to break immune tolerance.

[0186] a. Design history Each peptide immunogen construct or immunotherapy product requires its own unique design focus and approach based on its specific disease mechanism and target protein(s) requiring intervention. For the treatment of patients with PCSK9-mediated disorders, including elevated serum levels of low-density lipoprotein cholesterol (LDL-C) and CV events, PCSK9 was selected as the target molecule based on available scientific information, as outlined in Figures 1–4. The path from discovery to commercialization depicted in Figure 1 typically takes more than 10 years to achieve. To design an immunogen construct, it is crucial to identify the PCSK9 B-cell epitope peptide associated with the functional site(s) of interest. Serial pilot immunogenicity studies were conducted in guinea pigs incorporating various helper T-cell supports (carrier proteins or appropriate helper T-cell peptides) into various formulations, followed by evaluation of the functional properties of purified induced antibodies or vaccine formulations using specific PCSK9 peptide immunogen constructs in specific in vitro functional assays or proof-of-concept in vivo studies in selected animal models. Upon extensive serological validation, candidate PCSK9 B-cell epitope peptide immunogen constructs can be further tested in non-human primates to further validate the immunogenicity and directionality of the PCSK9 peptide immunogen design. Selected PCSK9 peptide immunogen constructs can then be prepared as different mixtures to evaluate subtle differences in the functional properties associated with the respective interactions between the peptide constructs when used in combination. Upon further evaluation, the final peptide constructs, peptide compositions, and pharmaceutical formulations thereof, along with the respective physical parameters of such formulations, can be established to lead to the final product development process.

[0187] The amino acid sequences of the PCSK9 peptide immunogen constructs were selected based on a number of design rationales. Some of these rationales include: (i) Use of a PCSK9 B cell epitope peptide sequence that does not contain a self-helper T cell epitope in PCSK9, thereby preventing autologous T cell activation; (ii) use of a PCSK9 B cell epitope peptide sequence that is not a self molecule and therefore is non-immunogenic by itself; (iii) using a PCSK9 B-cell epitope peptide sequence that can be made immunogenic by a protein carrier or a potent helper T-cell epitope(s) upon administration to a host; (iv) using PCSK9 B cell epitope peptide sequences that induce high titer antibodies directed against the PCSK9 peptide sequence (B cell epitope) but not against the protein carrier or strong helper T cell epitope(s); (v) using a PCSK9 B-cell epitope peptide sequence that enables efficient LDL-C uptake by LDL-R-expressing cell lines as measured by an in vitro LDL-C uptake assay by eliciting high-titer antibodies that inhibit / inhibit PCSK9 binding to LDL-R on LDL-R-expressing cell lines; and (vi) Such vaccine formulations include those employing PCSK9 B-cell epitope peptide sequences that, when administered to an animal, result in a time-dependent reduction in plasma / serum levels of LDL-C and T-CHO in the vaccinated animal.

[0188] b. Design and validation of PCSK9 peptide immunogen constructs for pharmaceutical compositions with the potential to treat patients susceptible to or suffering from PCSK9-mediated disorders, including elevated serum levels of low-density lipoprotein cholesterol (LDL-C) and CV events. To obtain the most potent peptide constructs for inclusion in pharmaceutical compositions, a repertoire of human PCSK9 B cell epitope peptides (e.g., SEQ ID NOS: 2-9) and nonselective or artificial helper T cell epitopes derived from various pathogens (e.g., SEQ ID NOS: 13-64) was designed. A number of PCSK9 peptide immunogen constructs (e.g., SEQ ID NOS: 65-107) were prepared for immunogenicity testing (initially in guinea pigs).

[0189] i) Selection of PCSK9 B-cell epitope peptide sequences from the receptor binding region or receptor activation region for design As shown in Figure 3, the interface residues between PCSK9 and the LDL-R receptor are located within the catalytic domain of the PCSK9 molecule, at amino acid sequences around 153-172, 211-223, and 368-382, respectively. The catalytic domain of PCSK9 around these regions was selected for PCSK9 B cell epitope design, and then PCSK9 peptide immunogen constructs were further generated, as shown in Table 3. The peptide immunogen constructs were then used to elicit immune sera in guinea pigs for immunogenicity evaluation by ELISA on PCSK9 B cell epitope peptide-coated plates, followed by in vitro functional assay evaluation.

[0190] Normally, LDL-C bound to LDL-R is internalized into hepatocytes via clathrin-coated vesicles, after which the acidic environment of the endosome dissociates LDL-C from its receptor. While recycling vesicles return LDL-R to the cell surface, endosomes containing LDL-C particles fuse with lysosomes, resulting in LDL-C degradation, cholesterol ester hydrolysis, and distribution of free cholesterol to other cellular compartments. At the plasma membrane of hepatocytes, the catalytic domain of secreted PCSK9 associates with LDL-R, is internalized, and enters the endosomal pathway. The low pH of the endosome enhances the affinity of PCSK9 for LDL-R, preventing the receptor from recycling to the cell surface. Instead, the complex is targeted to lysosomes, where both components are degraded. Additionally, PCSK9 may complex with LDL-R in the Golgi apparatus and target the receptor to lysosomes for degradation instead of transport to the plasma membrane, potentially enhancing intracellular LDL-R degradation before secretion.

[0191] Representative PCSK9 B-cell epitopes, such as those having SEQ ID NOS: 2-9, as shown in Figure 5, and their corresponding PCSK9 peptide immunogen constructs (e.g., SEQ ID NOS: 65-76) were designed and synthesized. Formulations containing these PCSK9 peptide immunogen constructs were administered to guinea pigs for the purpose of immunizing and eliciting potent polyclonal antibodies that target PCSK9 B-cell epitopes and cross-react with the full-length PCSK9 protein.

[0192] Two cysteine ​​residues are present within the PCSK9 B cell epitope region (SEQ ID NO: 5) at amino acids 368-382, forming a small four-member loop / ring constrained by Cys-Cys interactions. Another modified version of this B cell epitope was designed for immunogenicity and functional testing. Specifically, the native cysteine ​​residues (at aa 375 and aa 378) were replaced with serine residues, and the N-terminal isoleucine (at aa 368) and C-terminal glutamine (at aa 382) were replaced with cysteine ​​residues to create the modified B cell epitope sequence of SEQ ID NO: 6. The modified B cell epitope sequence of SEQ ID NO: 6 forms a larger 15-member loop / ring structure constrained by Cys-Cys interactions, which mimics the microenvironment surrounding the C-terminal LDL-R receptor binding region.

[0193] For the PCSK9 B-cell epitope region from amino acids 211 to 223, the glutamic acid (Glu) amino acid at amino acid position 211 was replaced with a cysteine ​​residue. As a result, the modified B-cell epitope sequence of SEQ ID NO: 7 forms a 13-member Cys-Cys loop / ring between the cysteine ​​at aa 211 and the native cysteine ​​at aa 223, which mimics the microenvironment around the C-terminal LDL-R receptor binding region.

[0194] PCSK9 peptide immunogen constructs containing SEQ ID NOs: 2-9 were initially formulated for immunogenicity studies with ISA51 and CpG for a prime immunization in guinea pigs at 400 μg / 1 mL and boosts at 100 μg / 0.25 mL (3 wpi, 6 wpi, and 9 wpi).

[0195] To test immunogenicity in guinea pigs, ELISA assays were performed using guinea pig immune sera (subjected to 10-fold serial dilutions from 1:100 to 1:10,000) obtained from blood samples at various time points (wpi). ELISA plates were coated with the corresponding human and guinea pig PCSK9 B cell epitope peptides and full-length PCSK9 protein at 0.5 μg of peptide per well. 450 The titer of the test serum (Log ) was calculated by linear regression analysis of A450nm with a cutoff value of 0.5. 10 The corresponding immunoreactivity with PCSK9 B cell epitope peptides (SEQ ID NOS: 4, 5, 6, and 7) is shown in Table 4 and Figure 6, with detailed titers of representative B cell epitope-derived PCSK9 peptide immunogen constructs. Although designed short PCSK9 peptides are often non-immunogenic due to the absence of endogenous Th epitopes, the addition of exogenous Th epitopes enhanced the immunogenicity of certain PCSK9 peptide immunogen constructs.

[0196] Further evaluation of the analysis of the reactivity / specificity patterns of the various constructs reported in Table 4 and Figure 6 may facilitate the design of optimal peptide immunogen constructs. Cross-reactivity with full-length PCSK9 protein from guinea pig immune sera collected over various time points was also evaluated for their ability to function in biological systems. High cross-reactivity with full-length recombinant human PCSK9 protein was confirmed at most, if not all, time points, as shown in Figure 7, along with the Log 10 EC 50 The antibody titers were confirmed by immunoassay using the PCSK9 peptide constructs. Such high cross-reactivity observed in these immune sera between the short PCSK9 B cell epitope peptides and the epitope peptides of the full-length PCSK9 protein demonstrates the high precision / fidelity nature of the immunogen design of the disclosed PCSK9 peptide immunogen constructs.

[0197] Table 5 provides a ranking of the binding efficiency of antibodies generated by the PCSK9 peptide immunogen constructs (SEQ ID NOS: 65 and 68-76) to rPCSK9 protein based on the results shown in FIG.

[0198] ii) The self-helper T cell epitope is not present within the selected PCSK9 B cell epitope Representative PCSK9 B cell epitopes tested, including those containing SEQ ID NOs: 2 and 3, did not elicit any antibodies to PCSK9 (data not shown). These results demonstrate that the PCSK9 B cell epitopes described herein do not contain undesirable endogenous Th epitopes that can themselves elicit an immune response.

[0199] iii) The antibody response elicited by the PCSK9 peptide immunogen construct targets only PCSK9 B cell epitopes and not Th epitopes Although all of the carrier proteins used to enhance immune responses directed against target B cell epitope peptides (e.g., keyhole limpet hemocyanin (KLH) protein, diphtheria toxoid (DT) protein, and tetanus toxoid (TT) protein) induce antibodies in immunized hosts when such B cell epitope peptides are chemically conjugated to their respective carrier proteins, more than 90% of these antibodies are directed against the enhancing carrier protein, and less than 10% are directed against the target B cell epitope.

[0200] Therefore, it was interesting to evaluate the specificity of the PCSK9 peptide immunogen constructs of the present invention. One representative PCSK9 peptide immunogen construct (SEQ ID NO: 65) was prepared for immunogenicity evaluation, containing the human PCSK9 153-162 B cell epitope linked via a spacer sequence to the heterologous T cell epitope UBITh®1 (SEQ ID NO: 37). UBITh®1 (the helper T cell peptide used for B cell epitope boosting) was coated on an ELISA plate, and guinea pig immune sera were evaluated to test for cross-reactivity with the UBITh®1 peptide used for boosting. Results demonstrated that, in contrast to the high immunogenicity of these constructs against the corresponding target PCSK9 B cell epitope peptide, the immune sera were found to be non-reactive against the UBITh®1 peptide (data not shown).

[0201] In summary, peptide immunogen design incorporating a target PCSK9 B cell epitope peptide linked to a carefully selected helper T cell epitope allows for the generation of a focused immune response that targets only the corresponding PCSK9 B cell epitope peptide.Based on the obtained data, it has been found that pharmaceutical compositions that generate a highly specific immune response directed against PCSK9 B cell epitopes are consistent with a higher safety profile of the composition.Therefore, the PCSK9 peptide immunogen construct of the present disclosure is highly specific and extremely potent for its B cell target.

[0202] iv) Fine epitope mapping using immune sera directed against selected PCSK9 peptide immunogen constructs can be performed Detailed epitope mapping studies to assign antibody binding site(s) to specific residues within the target B-cell epitope region of PCSK9 can be performed by designing overlapping 10-mer peptides encompassing PCSK9 amino acids 144-182, 201-233, and 358-392, which encompass the PCSK9 and LDL-R receptor-binding regions of the catalytic domain of the PCSK9 molecule. These 10-mer peptides can be individually coated onto the wells of a 96-well microtiter plate as solid-phase immunoadsorbents. Pooled guinea pig antisera can be added at a 1:100 dilution in sample dilution buffer to plate wells coated with 2.0 μg / mL of the 10-mer peptide, followed by incubation at 37°C for 1 hour. After washing the plate wells with wash buffer, horseradish peroxidase-conjugated rProtein A / G can be added and incubated for 30 minutes. After another wash with PBS, substrate can be added to the wells and the absorbance at 450 nm can be measured using an ELISA plate reader. Samples are analyzed in duplicate. Maximal antibody binding signal will be indicated if PCSK9 peptide immunogen-elicited immune serum binds to wells coated with the corresponding PCSK9 B-cell epitope peptide.

[0203] Detailed epitope mapping results will reveal whether pooled guinea pig sera from PCSK9 peptide immunogen constructs containing PCSK9 B cell epitope peptides derived from the N-terminal, central, and / or C-terminal regions of the catalytic domain induce high-titer antibodies with high cross-reactivity to recombinant human PCSK9 protein compared to other B cell epitope peptides.

[0204] In summary, the designed synthetic PCSK9 peptide immunogen constructs tested to date induced robust immune responses in guinea pigs and generated polyclonal antibodies targeting distinct clusters of B cell epitope peptides within the catalytic domain of the PCSK9 molecule. These regions are in close proximity to the PCSK9-LDL-R receptor binding region near the N-terminal, central, and C-terminal regions of each of the catalytic domains of the PCSK9 molecule, allowing for important medical interventions. Epitope mapping, coupled with functional assay evaluation, will enable the identification of optimal peptide immunogen constructs for use in pharmaceutical compositions containing the PCSK9 peptide immunogen constructs.

[0205] Example 7 Functional characterization of antibodies by measuring in vivo serum / plasma levels of LDL-C and T-CHO in hosts immunized with PCSK9 peptide immunogen constructs and their formulations After demonstrating the high immunogenicity and cross-reactivity of immune sera from guinea pigs immunized with carefully selected candidate PCSK9 immunogen constructs, as shown in Table 4 and Figures 6 and 7, the following study was designed to evaluate whether serum / plasma levels of LDL-C and T-CHO were affected as a result of immunization of guinea pigs, given the close sequence identity between guinea pig and human sequences.

[0206] Measurement of in vivo serum / plasma levels of LDL-C and T-CHO in guinea pigs immunized with PCSK9 peptide immunogen constructs As described above in Example 3, serum / plasma levels of LDL-cholesterol (LDL-C) and total cholesterol (T-CHO) were measured for each blood sample from each animal using a Hitachi 7080 analyzer with a Wako L-type CHO M kit (catalog number 462-12491) and a Roche L-type LDL-C kit (catalog number 137520), respectively, according to the manufacturer's instructions. Dilutions of T-CHO / LDL-C standards or test samples (70 μL per sample volume) were added to each well of a 96-well plate. 140 μL of prepared LDL-C reagent was added as a calibrator. The plate was incubated at 37°C for 5 minutes, and the absorbance of the developed color was read at 600 nm within 30 minutes. Figures 8A-8B and 9A-9B show LDL-C and T-CHO levels, respectively, measured in guinea pigs immunized with PCSK9 peptide immunogen constructs (SEQ ID NOS: 65, 66, 68, 75, and 68-74) bearing PCSK9 B-cell epitopes derived from the N-terminal, central, and C-terminal regions within the catalytic domain of PCSK9. Significant reductions in serum / plasma LDL-C were found for constructs bearing B-cell epitopes derived from both the N-terminal and C-terminal regions as early as week 3 after the first immunization at week 0, as shown in the left panels of Figures 8A and 8B. Such reductions were approximately 20-50% when compared to basal LDL-C levels measured at week 0. All LDL-C levels were further compared to those in the placebo group. Overall reductions ranged from 30-50% when compared to levels in placebo animals at each time point. A delayed decrease in LDL-C levels was observed in animals immunized with the PCSK9 immunogen construct of SEQ ID NO: 75, which contains a B cell epitope derived from the central region of the PCSK9 catalytic domain (SEQ ID NO: 7), over the first 9 weeks, despite reasonable antibody cross-reactivity with the full-length recombinant PCSK9 protein. The rate of LDL-C reduction then returned to levels comparable to those of the N- and C-terminal regions of the PCSK9 B cell epitope. As shown in Figures 9A and 9B, a parallel trend in the decrease in T-CHO serum / plasma levels was observed for all serum / plasma samples collected at all measured time points.

[0207] In summary, we observed immediate and significant reductions in serum / plasma levels of LDL-C and T-CHO in all animals immunized with the specially designed PCSK9 peptide immunogen constructs, demonstrating that the PCSK9 peptide immunogen constructs and formulations containing the constructs of the present disclosure have in vivo efficacy evidence. These results demonstrate that the PCSK9 peptide immunogen constructs can break immune tolerance by generating site-specific antibodies against critical self-proteins, thereby regulating LDL-C uptake by cells, such as hepatocytes, that express LDL-R, allowing for increased rates of LDL-C and T-CHO clearance from serum / plasma.

[0208] Table 5 provides a ranking of the % T-CHO and % LDL inhibition generated by antibodies elicited from PCSK9 peptide immunogen constructs (SEQ ID NOs: 65 and 68-76) based on the results shown in Figures 8A-8B and 9A-9B.

[0209] Example 8 Functional characterization of antibodies derived from hosts immunized with PCSK9 peptide immunogen constructs for their ability to enhance LDL-C uptake in an in vitro assay The functional properties of antibodies derived from hosts immunized with the disclosed PCSK9 peptide immunogen constructs and formulations thereof were evaluated by in vitro assays for LDL-C uptake, as described in Example 3, using LDL-R-expressing hepatocytes as a system for such measurements. Figure 10A illustrates the results of a representative study evaluating the ability of polyclonal antibodies purified from 12 wpi immune serum of guinea pigs immunized with PCSK9 peptide immunogen constructs (e.g., SEQ ID NOS: 65 and 75). The rationale for such study design is that anti-PCSK9 antibodies elicited by these peptide immunogen constructs inhibit and prevent PCSK9 binding to LDL-R. This inhibition would prevent internalization of the PCSK9-LDL-R complex and subsequent cellular events leading to LDL-R degradation. Reduced surface expression of LDL-R due to receptor recycling would reduce its uptake of LDL-C from serum / plasma. The procedure for the LDL uptake assay is shown in the left panel of Figure 10A, and the LDL uptake rate results (indicating surface LDL-R expression that is not degraded by PCSK9 binding) are shown as a bar graph on the right panel. As can be seen from Figure 10A, using purified antibodies from both PCSK9 peptide immunogen constructs having SEQ ID NOs: 65 and 75, an antibody dose (0, 5, 15, 100, 500, 1,000, 1,250 μg / mL)-dependent enhancement of LDL-C uptake is observed.

[0210] Similar studies were performed using the assay procedure shown in Figure 10A with polyclonal antibodies purified from immune serum 12 wpi of guinea pigs immunized with peptide immunogen constructs of the N-terminal region (SEQ ID NO: 65), central region (SEQ ID NOs: 75-76), and C-terminal region (SEQ ID NOs: 68-74) of PCSK9 at antibody doses ranging from 0, 50, 250, and 500 μg / mL, as shown in Figure 10B. Figure 10B shows that antibody dose-dependent enhancement of LDL-C uptake was observed using purified antibodies from SEQ ID NOs: 65 and 75, which is consistent with the results shown in Figure 10A. Figure 10B also shows that PCSK9 peptide immunogen constructs of SEQ ID NOs: 71 and 76 also produced dose-dependent enhancement of LDL-C uptake, with SEQ ID NO: 75 producing a significantly stronger response than SEQ ID NO: 71. Interestingly, all of the PCSK9 peptide immunogen constructs of SEQ ID NOs: 68-70 and 72-74 resulted in enhanced LDL-C uptake at an antibody dose of 50 μg / mL, but the enhancement was reduced at higher antibody concentrations (250 and 500 μg / mL).

[0211] A third study was performed using the assay procedure shown in Figure 10A with polyclonal antibodies purified from immune serum 15 wpi from guinea pigs immunized with peptide immunogen constructs of the N-terminal region (SEQ ID NO: 65), central region (SEQ ID NOs: 75-76), and C-terminal region (SEQ ID NO: 70) of PCSK9 at antibody doses ranging from 1,250, 1,000, 500, 100, 15, 5, and 0 μg / mL, as shown in Figure 10C. Figure 10C shows that antibody dose-dependent enhancement of LDL-C uptake was observed using purified antibodies from SEQ ID NOs: 65 and 75, which is consistent with the results shown in Figures 10A and 10B. The results for SEQ ID NO: 75 indicate that the level of LDL-C uptake enhancement was relatively constant across the antibody dose range of 5 to 1,000 μg / mL. The results for SEQ ID NO: 76 demonstrate that an antibody dose of 500 μg / mL resulted in the strongest LDL-C uptake compared to other doses. Finally, the results for SEQ ID NO: 70 demonstrate that antibody doses of 5 and 500 μg / mL result in the strongest LDL-C uptake compared to the other doses tested.

[0212] In summary, purified antibodies derived from the PCSK9 and LDL-R binding regions, from immune sera at 12 wpi from guinea pigs immunized with representative PCSK9 peptide immunogen constructs of SEQ ID NOS: 65, 75, 76, and 68-74 and from immune sera at 15 wpi from guinea pigs immunized with SEQ ID NOS: 65, 75, 76, and 70, demonstrate important functional properties of anti-PCSK9 polyclonal antibodies that confer in vivo efficacy by enhancing LDL-R uptake in cells expressing the LDL-R, resulting in better clearance of LDL-C from circulating serum / plasma.

[0213] Table 5 provides a ranking of the percent LDL uptake generated by antibodies elicited from PCSK9 peptide immunogen constructs (SEQ ID NOS: 65 and 68-76) based on the results shown in Figures 10A-10C.

[0214] Example 9 Evaluation of immunogenicity and functional properties of antibodies directed against the N-terminal region of PCSK9 The immunogenicity of two additional PCSK9 peptide immunogen constructs, SEQ ID NOs: 66 and 67, containing the B-cell epitopes of SEQ ID NOs: 3 and 4, respectively, was evaluated according to the protocol described in Example 6.

[0215] The left panel of Figure 11 shows that guinea pigs immunized with the PCSK9 peptide immunogens of SEQ ID NO: 66 or 67 produced high immunogenic titers starting as early as 3 wpi, and these titers were maintained up to 15 wpi.

[0216] The graph shown in the right panel of Figure 11 demonstrates that antibodies elicited by the peptide immunogen constructs were highly reactive to the B cell epitopes of PCSK9 (SEQ ID NOs: 3 and 4) but not to the Th epitopes of UBITh®1 or the CpG3 oligonucleotide.

[0217] In addition, serum T-CHO and LDL-C levels were evaluated in animals immunized with the PCSK9 peptide immunogen constructs of SEQ ID NOs: 66 and 67. Specifically, guinea pigs were immunized with the PCSK9 peptide immunogen according to the protocol described in Table 6.

[0218] The results of this study are shown in Figure 12, with the results for SEQ ID NO: 65 (shown in Figures 8A and 9A) included for comparison. Specifically, Figure 12 shows that peptide immunogen constructs from the N-terminal region of PCSK9 (SEQ ID NOs: 65-67) were effective in reducing T-CHO and LDL levels in immunized guinea pigs. The levels of T-CHO and LDL reduction were comparable among SEQ ID NOs: 66-67 throughout the study, with T-CHO and LDL levels being significantly reduced in guinea pigs immunized with SEQ ID NO: 65 compared to SEQ ID NOs: 66 and 67.

[0219] [Table 1]

[0220] [Table 2]

[0221] [Table 3]

[0222] [Table 4]

[0223] [Table 5]

[0224] [Table 6]

[0225] Table 7

[0226] Table 8

[0227] Table 9

Claims

1. The following formula: (Th) ｍ -(A) ｎ -(PCSK9 functional B cell epitope peptide)-X or (PCSK9 functional B cell epitope peptide)-(A) ｎ - (Th) ｍ -X or (Th) ｍ -(A) ｎ -(PCSK9 functional B cell epitope peptide)-(A) ｎ - (Th) ｍ -X is expressed as During the ceremony, Th is a heterologous helper T cell epitope of SEQ ID NO: 37; A is a heterologous spacer which is (α,ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 11), or Lys-Lys-Lys-ε-N-Lys (SEQ ID NO: 12); (PCSK9 functional B-cell epitope peptide) is a B-cell epitope peptide selected from the group consisting of SEQ ID NOs: 2 to 7, X is the α-COOH or α-CONH of an amino acid ２ and m is 1 to 4; A PCSK9 peptide immunogen construct, wherein n is 1-10.

2. The PCSK9 peptide immunogen construct of claim 1, wherein the PCSK9 functional B-cell epitope peptide is the amino acid sequence of SEQ ID NO:

2.

3. The PCSK9 peptide immunogen construct of claim 1, wherein the PCSK9 functional B-cell epitope peptide is the amino acid sequence of SEQ ID NO:

7.

4. 2. The PCSK9 peptide immunogen construct of claim 1, wherein n is 1 and the heterologous spacer is selected from the group consisting of ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 11) and Lys-Lys-Lys-ε-N-Lys (SEQ ID NO: 12).

5. 2. The PCSK9 peptide immunogen construct of claim 1, wherein n is 1 and m is 1.

6. 2. The PCSK9 peptide immunogenic construct of claim 1, wherein said peptide immunogenic construct is selected from the group consisting of SEQ ID NOs: 65-76.

7. 2. The PCSK9 peptide immunogenic construct of claim 1, wherein the peptide immunogenic construct is selected from the group consisting of SEQ ID NOs: 65, 66, 67, 70, 71, 75 and 76.

8. 2. The PCSK9 peptide immunogenic construct of claim 1, wherein the peptide immunogenic construct is selected from the group consisting of SEQ ID NOs: 65, 75 and 76.

9. a. a B-cell epitope selected from the group consisting of SEQ ID NOs: 2-7; b. a helper T cell epitope of SEQ ID NO: 37; and c. a heterologous spacer selected from the group consisting of (α,ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 11), and Lys-Lys-Lys-ε-N-Lys (SEQ ID NO: 12); A PCSK9 peptide immunogen construct comprising: The PCSK9 peptide immunogen construct, wherein the B cell epitope is covalently linked to the helper T cell epitope via the heterologous spacer.

10. 10. The PCSK9 peptide immunogen construct of claim 9, wherein the B-cell epitope is selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:

7.

11. 10. The PCSK9 peptide immunogen construct of claim 9, wherein the B-cell epitope is selected from the group consisting of SEQ ID NO:5 and SEQ ID NO:

6.

12. 10. The PCSK9 peptide immunogen construct of claim 9, wherein the helper T cell epitope is covalently linked to the amino or carboxyl terminus of the B cell epitope via the heterologous spacer, and the heterologous spacer is ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 11) or Lys-Lys-Lys-ε-N-Lys (SEQ ID NO: 12).

13. 10. The PCSK9 peptide immunogenic construct of claim 9, wherein the peptide immunogenic construct is SEQ ID NO:

65.

14. A composition comprising the PCSK9 peptide immunogenic construct of any one of claims 1 to 13.

15. a. a PCSK9 peptide immunogenic construct according to any one of claims 1 to 13; and b. a pharmaceutically acceptable delivery vehicle and / or adjuvant; A pharmaceutical composition comprising:

16. 16. The pharmaceutical composition of claim 15, wherein the PCSK9 peptide immunogen construct is mixed with a CpG oligodeoxynucleotide (ODN) to form a stabilized immunostimulatory complex.

17. 17. The pharmaceutical composition of claim 16, wherein the PCSK9 peptide immunogenic construct is selected from the group consisting of SEQ ID NOs: 65-76.

18. 18. The pharmaceutical composition of claim 17, wherein the PCSK9 peptide immunogenic construct is selected from the group consisting of SEQ ID NOs: 65, 75 and 76.

19. 14. Use of the PCSK9 peptide immunogenic construct of any one of claims 1 to 13 in the manufacture of a medicament for generating antibodies against PCSK9 in a patient.

20. 16. The pharmaceutical composition of claim 15, which generates antibodies against PCSK9 in a patient.

21. An isolated antibody elicited by the PCSK9 peptide immunogenic construct of any one of claims 1 to 13, or an epitope-binding fragment thereof that specifically binds to a B-cell epitope peptide of any one of SEQ ID NOs: 2 to 7, wherein the isolated antibody or the epitope-binding fragment binds to the PCSK9 peptide immunogenic construct of any one of claims 1 to 13.

22. 22. A composition comprising the isolated antibody or epitope-binding fragment thereof of claim 21.

23. Use of the PCSK9 peptide immunogenic construct of any one of claims 1 to 13 in the manufacture of a medicament for preventing and / or treating a PCSK9-mediated disorder in a patient.

24. 24. The use of claim 23, wherein the PCSK9-mediated disorder is increased serum levels of low-density lipoprotein cholesterol (LDL-C).

25. 16. The pharmaceutical composition of claim 15, for preventing and / or treating a PCSK9-mediated disorder in a patient.

26. 26. The pharmaceutical composition of claim 25, wherein the PCSK9-mediated disorder is increased serum levels of low-density lipoprotein cholesterol (LDL-C).

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